Journal: eLife

Article Title: A vibrissa pathway that activates the limbic system

doi: 10.7554/eLife.72096

Figure Lengend Snippet: ( A ) Viral method used for labeling paralemniscal projections. ( B ) Labeling of interpolaris cells after injection of G-pseudotyped Lenti-Cre virus in Po thalamus, and a Cre-dependent AAV that expresses GFP in the vibrissa-responsive sector of SpVIr. Horizontal section. ( C ) Anterograde labeling of terminal fields in Po thalamus and zona incerta. Sagittal section. ( D ) Anterograde labeling in the KF/PBc. Horizontal section. ( E ) Anterograde labeling in the ITr. Horizontal section. ( F ) Anterograde labeling in the dorsal sector of the facial nucleus. Horizontal section. ( G ) Anterograde labeling in the MdD and cervical cord. Horizontal section. ( H ) Population peristimulus time histogram of spike discharges evoked in KF (22 cells) by air puff deflection of the vibrissae in the anesthetized rat. A representative response is shown in the insert. ( I ) Example of a vibrissa responsive KF cell labeled by juxtacellular delivery of Neurobiotin. Horizontal section. ( J ) Location of eight juxtacellularly labeled KF cells. Horizontal brainstem sections in ( I ) and ( J ) were counterstained for cytochrome oxidase. Horizontal section. ( K ) Spectral coherence of spontaneous discharges of KF cells with the respiratory cycle at the respiratory frequency; 1–3 Hz. Note that, in contrast with the respiratory units (blue dots), spontaneous discharges of vibrissa-responsive cells (red triangles) display low coherence with respiration. ( L ) Population peristimulus time histogram of spike discharges evoked in MdD (33 cells) by air puff deflection of the vibrissae. ( M ) Recording site in the MdD labeled by an iontophoretic injection of Chicago Sky Blue. This coronal section was counterstained for cytochrome oxidase and a negative image was generated. Coronal section. See for additional anatomical data. Abbreviations for all anatomy: 5n, root of the trigeminal motor nucleus; 5N, trigeminal motor nucleus; 5t, trigeminal tract; 7n, facial nerve tract; 7N, facial nucleus; Amb, ambiguus nucleus; APT, anterior pretectal nucleus; BSTL, bed nucleus of the stria terminalis; CeA, central amygdala; Cerv Cord, cervical cord; CM/PC, central medial/paracentral thalamic nuclei; CPu, caudate putamen; DR, dorsal raphe; EW, Edinger-Westphal; Hab, habenula; IML, intermedio-lateral column of the spinal cord; ITr, intertrigeminal region; KF, Kölliker-Fuse nucleus; KF/PBc, Kölliker-Fuse/parabrachial complex; mcp, middle cerebellar peduncle; MdD, dorsal part of the medullary reticular formation; MdV, ventral part of the medullary reticular formation; mt, mammillothalamic tract; NA, nucleus ammbiguus; NTS, nucleus of the solitary tract; opt, optic tract; PAG, periaqueductal gray; PB, parabrachial nuclei; PC, paracentral thalamic nucleus; PCRt, parvicellular reticular formation; PLH, posterior lateral hypothalamus; Po, posterior nuclear group of the thalamus; PrV, principal trigeminal nucleus; RN, red nucleus; Rt, reticular thalamic nucleus; s5, sensory root of the trigeminal nerve; SC, superior colliculus; scp, superior cerebellar peduncle; SpVC, caudalis division of the spinal trigeminal complex; Sol, nucleus of the solitary tract; SpVIc, caudal sector of the interpolaris trigeminal nucleus; SpVIr, rostral division of the interpolaris nucleus; TG, trigeminal ganglion; VLL, ventral nucleus of the lateral lemniscus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posterior medial nucleus; VPCc, parvicellular sector of the ventral posteromedial thalamic nucleus; VPPc, parvocellular part of the ventral posterior thalamic nucleus; VRG, ventral respiratory group; vsc, ventral spinocerebellar tract; ZIv, ventral division of zona incerta.

Article Snippet: For brightfield microscopy, sections were first counterstained for cytochrome oxidase (MilliporeSigma), and then immunoreacted with a rabbit anti-GFP antibody (1:1000; Novus Biological), a biotinylated horse anti-rabbit IgG (1:200; Vector Labs), the avidin/biotin complex (Vectastain ABC Kit; Vector Labs), and the SG peroxidase substrate (ImmPACT SG Substrate; Vector Labs).

Techniques: Labeling, Injection, Virus, Generated

Journal: eLife

Article Title: A vibrissa pathway that activates the limbic system

doi: 10.7554/eLife.72096

Figure Lengend Snippet:

Article Snippet: For brightfield microscopy, sections were first counterstained for cytochrome oxidase (MilliporeSigma), and then immunoreacted with a rabbit anti-GFP antibody (1:1000; Novus Biological), a biotinylated horse anti-rabbit IgG (1:200; Vector Labs), the avidin/biotin complex (Vectastain ABC Kit; Vector Labs), and the SG peroxidase substrate (ImmPACT SG Substrate; Vector Labs).

Techniques: Plasmid Preparation, Recombinant, Virus, Software

Journal: Journal of molecular cell biology

Article Title: Dazl promotes germ cell differentiation from embryonic stem cells.

doi: 10.1093/jmcb/mjp026

Figure Lengend Snippet: Figure 5 Suppressed Nanog expression and induced GCNA expression in Dazl-transfected mES cells. Mouse ES cells were transiently trans- fected with plenti6Ubc-Dazl-V5, by which Dazl is expressed as a fusion protein with the V5 tag, and cultured under standard ES medium with LIF for 24 h. (A, B) Dual-immunofluorescence staining of V5 and Nanog shows that Nanog expression was suppressed in Dazl-overexpressing mES cells. (C and D) Dual-immunofluorescence staining of GCNA and Nanog. (E–H) Dual-immunofluorescence staining of V5 and GCNA, G is the overlap of E and F, H is the overlap of V5, GCNA and DAPI; this result indicates that the expression of GCNA was induced by Dazl.

Article Snippet: For immunofluorescence with cells, the following antibodies were used: FE-J1 antibody (The Developmental Studies Hybridoma Bank, University of Iowa); acetyl-a-tubulin antibody (Sigma-Aldrich Corp, St. Louis, MO, USA); Anti-CDF-9 antibody (R&D Systems Inc., Minneapolis, MN 55413, USA); Oct4 antibody (c-10 monoclonal ab from Santa Cruz Biotech, CA, USA); V5 antibody (Invitrogen); Nanog antibody (R&D Systems); GCNA antibody (Rat IgG, kindly provided by George C. Enders, The University of Kansas); Stra8 antibody (Abcam, Cambridge, MA, USA); b-Tubulin (Sigma, Clone Tub2.1).

Techniques: Expressing, Transfection, Cell Culture, Staining

Journal: Journal of molecular cell biology

Article Title: Dazl promotes germ cell differentiation from embryonic stem cells.

doi: 10.1093/jmcb/mjp026

Figure Lengend Snippet: Figure 6 Dazl regulates the expression of germ cell specific genes. (A) Mouse ES cells were infected by plenti6Ubc-Dazl-V5 lentivirus medium for the overexpression of Dazl. Western blotting analyses shows that Dazl was overexpressed and that overexpression of Dazl induced the expression of GCNA (top panel, control was of mES cells without transfection), whereas the expression of Nanog was suppressed by Dazl (bottom panel). (B) Western blotting analysis shows that Dazl was knocked down by siRNA in mES cells; Dazl siRNA2 was more efficient than Dazl siRNA1, thus, the cells trans- fected with Dazl siRNA2 were used for further experiments. (C) RT– PCR analyses were performed to test the expression of early germ cell markers including Stella, MVH and Prdm1, in the Dazl knockdown mES cells. (D) RT–PCR analyses show that germ cell markers includ- ing Prdm1 for PGCs, Stra8 for premeiotic germ cells and Protamine1 for postmeiotic cells, were activated temporally in Dazl-transfected mES cells cultured under DMEM/F12 medium containing 15% FBS without LIF. (Con/Ve is for mixed total RNAs collected from non- infected cells and vector-infected cells cultured over the same period of culture time as did for pLenti-UBC-Dazl-V5 infected cells, Day 1 is for 1 day after infection, Day 2 is for 2 days after infection and so on).

Article Snippet: For immunofluorescence with cells, the following antibodies were used: FE-J1 antibody (The Developmental Studies Hybridoma Bank, University of Iowa); acetyl-a-tubulin antibody (Sigma-Aldrich Corp, St. Louis, MO, USA); Anti-CDF-9 antibody (R&D Systems Inc., Minneapolis, MN 55413, USA); Oct4 antibody (c-10 monoclonal ab from Santa Cruz Biotech, CA, USA); V5 antibody (Invitrogen); Nanog antibody (R&D Systems); GCNA antibody (Rat IgG, kindly provided by George C. Enders, The University of Kansas); Stra8 antibody (Abcam, Cambridge, MA, USA); b-Tubulin (Sigma, Clone Tub2.1).

Techniques: Expressing, Infection, Over Expression, Western Blot, Control, Transfection, Reverse Transcription Polymerase Chain Reaction, Knockdown, Cell Culture, Plasmid Preparation

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Immunoblotting images showing the protein levels of HIF1α and its targets in 9 types of normal and cancer cells under normoxia (N) and hypoxia (H; 0.2% O 2 for 24 hours). n = 5-8. b, Confocal microscopy shows HIF1α expression in aerobic cell cultures of human PASMCs, PAECs, and AoECs. n = 3. c, HIF1α and α-smooth muscle actin (αSMA, a marker of VSMCs) expression in human lung tissues of transplantation-failed donors. n = 3. d, Glycolytic extracellular acidification rate (ECAR) in normoxic cell cultures determined by a Seahorse glycolytic stress assay. n = 5-9. e,f, PFKFB3 ( e ) and LDHA ( f ) protein levels in cells cultured in 21% or 0.2% O 2 for 24 hours. Fold change was calculated relative to corresponding normoxic cultures of each cell type. n = 5-8. g, Extracellular lactate levels in different normal and cancer cell types under normoxia or hypoxia (0.2% O 2 ) for 24 hours. n = 4-5. h, mRNA expression of HIF1α and its known transcriptional genes in aerobic cultures of PASMCs transfected with control siRNA (siCtrl) or HIF1α siRNA (siHIF1α). n = 9. i,j, Seahorse assays show ECAR ( i ) and oxygen consumption rate (OCR; j ) in PASMCs with HIF1α knockdown under normoxia. n = 3. k, Lactate secretion by PASMCs with HIF1α knockdown. Fold change was calculated relative to siCtrl-transfected cells. n = 7. All data were presented as mean ± SD. One-way ANOVA followed by Dunnett’s post-hoc test ( d ), Student’s t test or Mann-Whitney U test ( e-k ) was used when compared to aerobic culture of PASMCs or the matched cell type ( d-g ), or siCtrl-transfected PASMCs ( h-k ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Western Blot, Confocal Microscopy, Expressing, Marker, Transplantation Assay, Cell Culture, Transfection, Control, Knockdown, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Schematics shows medium conditioning and reapplication. b, Representative immunoblots of HIF1α and its regulatory proteins in PASMCs cultured in fresh growth medium (GM) or conditioned medium (CM). n = 3. c, Relative mRNA expression of HIF1α key target genes in glucose metabolism in PASMCs cultured in GM or CM. n = 7. d, Seahorse glycolytic stress test shows basal ECAR and glycolytic capacity in GM- or CM- cultured PASMCs. n = 5. e, Extracellular lactate levels in PASMCs grown in GM or CM. Fold change was calculated relative to GM-cultured cells. n = 10. f,g, Protein levels ( f ) and mRNA expression ( g ) of HIF1α and its transcriptional targets in PASMCs grown in GM or CM after transfection with control siRNA (siCtrl) or human HIF1α siRNA (siHIF1α). n = 3 ( f ) and 5 ( g ). h, LC-MS metabolomic profiling shows the levels of glycolytic metabolites G3P, pyruvate (PYR), and lactate (LAC) in PASMCs treated as described in panel f . n = 3. i, Seahorse glycolytic stress test shows basal ECAR and glycolytic capacity in CM-cultured PASMCs transfected with siCtrl or siHIF1α. n = 3. j, Extracellular lactate levels in GM or CM cultures of PASMCs with or without siHIF1α transfection. n = 5. All data are presented as mean ± SD. Student’s t test or Mann-Whitney U test ( c-e , i ), one-way ANOVA followed by Tukey’s post-hoc or Kruskal-Wallis test followed by Dunn’s post-hoc test ( g , h , j ) was used when compared to GM-cultured PASMCs ( c-e ), GM-cultured and siCtrl-transfected PASMCs ( g , h , j ), or siCtrl-transfected and CM-cultured PASMCs ( g-j ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Control, Liquid Chromatography with Mass Spectroscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Volcano plot of LC-MS metabolomic profiling shows the levels of 138 secreted metabolites in CM of PASMCs. n = 3. b, Quantitation of KIC/KMV (both have the same m/z ratio and are indistinguishable by LC-MS) and KIV in CM of PASMCs. ND: non-detectable. n = 3. c, Intracellular levels of KIC/KMV and KIV by LC-MS analysis in PASMCs cultured in GM or CM. Fold change was calculated relative to GM-cultured cells. n = 3. d, HPLC measurements of BCKAs in CM of PASMCs and basal GM. n = 3-6. e,f, Protein levels ( e ) and mRNA expression ( f ) of HIF1α and its target genes in PASMCs treated with KIC (100 μM), 50 μM of KMV or KIV, or all three BCKAs combined (KIC 100 μM, 50 μM KMV, and 50 μM KIV). n = 3 ( e ) and 5 ( f ). Fold change in f was calculated relative to vehicle control H 2 O treated cells. g, Protein levels of HIF1α, LDHA, and PDK1 in PASMCs cultured in GM or CM from PASMCs transfected with siRNAs for control (siCtrl), BCAT1 (siBCAT1), BCAT2 (siBCAT2 ) , or both BCAT1 and BCAT2 (siBCAT1/2). n = 3. h, HPLC determination of secreted BCKA levels in CM of PASMCs after transfection with siCtrl, siBCAT1, siBCAT2, or siBCAT1/2. n = 4. i-k, Representative immunoblotting images showing HIF1α and its target proteins PFKFB3 and LDHA in BCKA-treated human AoSMCs ( i ), CASMCs ( j ), and pericytes ( k ). n = 3. l, mRNA expression of HIF1α transcriptional genes in glycolysis in BCKA-treated human AoSMCs ( n = 4), CASMCs ( n = 5), and pericytes ( n = 6). Fold change was calculated relative to untreated control cells. All data are presented as mean ± SD. Student’s t test ( b-d ), one-way ANOVA followed by Dunnett’s test or Kruskal-Wallis test followed by Dunn’s test ( f , h , l) was applied when compared to GM ( b-d ), vehicle control H 2 O treated ( f , l ), or CM from siCtrl-transfected PASMCs ( h ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Cell Culture, Expressing, Control, Transfection, Western Blot

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Reaction rates of KIC (250 μM), KMV (250 μM), KIV (250 μM), and all three BCKAs (200 μM of each BCKAs) determined by a competitive inhibition assay. Roxadustat (Roxa; 100 μM) was included as a positive control. n = 4. b, Inhibition curve and IC 50 value of three BCKAs for PHD2 hydroxylase activity. n = 4. c, The predicted docking sites and energy of KIC, KMV, and KIV on PHD2 enzyme (crystal structure accession ID: 2G19). d, Schematic diagram shows the metabolic fates of BCKAs. e, Representative immunoblots in untreated and BCKA-treated PASMCs. n = 3. f, 13 C 6 -KIC tracing of the labelled leucine (Leu) in PASMCs with BCAT1 , BCAT2 , or both ( BCAT1/2 ) silencing. n = 6. g, LC-MS measurements of metabolite levels in BCKA-treated PASMCs. Fold change was calculated relative to untreated control cells. n = 6. h,i, Intracellular L2HG levels ( h ) and protein levels of HIF1α, PFKFB3, and LDHA ( i ) in siCtrl or LDHA siRNA (siLDHA) transfected PASMCs with or without BCKA treatment. n = 4 ( h ) and 3 ( i ). j, L2HGDH mRNA expression in siCtrl or L2HGDH siRNA (siL2HGDH) transfected PASMCs with or without BCKA treatment. n = 4. k,l, Intracellular L2HG levels ( k ) and protein levels of HIF1α, PFKFB3, and LDHA ( l ) in siCtrl or siL2HGDH transfected PASMCs with or without BCKAs. n = 4 ( k ) and 3 ( l ). m, Immunoblotting shows protein levels in cell permeable trifluoromethylbenzyl (TFMB) ester of L2HG (500 μM) or D2HG (500 μM) treated PASMCs. n = 3. n, Immunoblots of HIF1α, PFKFB3, and LDHA in siCtrl or HIF1α siRNA (siHIF1α) transfected PASMCs with or without cell permeable L2HG (TFMB-L2HG) (500 μM). n = 3. o, Proposed mechanisms of BCKA-induced inhibition of PHD2 activity. All data are presented as mean ± SD. One-way ANOVA followed by Dunnett’s test ( a , f ) or Tukey’s test ( h , j , k ), Student’s t test or Mann-Whitney U test ( g ) was used when compared to no inhibitor control ( a ), untreated control cells ( g ), siCtrl-transfected and control or BCKA-treated PASMCs ( f , h , j , k ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Inhibition, Positive Control, Activity Assay, Western Blot, Liquid Chromatography with Mass Spectroscopy, Control, Transfection, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, Volcano plot from LC-MS metabolomic profiling showing the changes of 136 metabolites in PASMCs with or without BCKA treatment. n = 6. b, Top 10 Metabolite Set Enrichment Analysis (MSEA) pathways in BCKA-treated PASMCs. c, LC-MS measurements of glycolytic metabolites in BCKA-treated PASMCs. Fold change was calculated relative to untreated controls. n = 6. d, Extracellular lactate levels in BCKA-treated PASMCs. Fold change was calculated relative to untreated controls. n = 4. e,f, Protein levels ( e ) and mRNA expression ( f ) of HIF1α and its transcriptional targets in PASMCs transfected with siCtrl or siHIF1α followed by stimulation with BCKAs. Fold change in f was calculated relative to control cells. n = 3 ( e ) and 4 ( f ). g, LC-MS measurements show the levels of glycolytic metabolites in PASMCs treated as described in panel e . Fold change was calculated relative to siCtrl-transfected and untreated controls. n = 3. h, Seahorse glycolytic stress test demonstrates glycolytic capacity of PASMCs treated as described in panel e . n = 3. i, Extracellular lactate levels in PASMCs treated as described in panel e . Fold change was calculated relative to siCtrl-transfected and untreated controls. n = 5. All data are presented as mean ± SD. Student’s t test or Mann-Whitney U test ( c , d ), or one-way ANOVA followed by Tukey’s test ( f-i ) was conducted when compared to untreated controls ( c , d ), siCtrl-transfected and untreated or BCKA-treated PASMCs ( f-i ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Transfection, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, mRNA expression of synthetic marker VIM and contractile marker ACTA2 in BCKAs (100 μM), TGFβ (2 ng/mL), or PDGF-BB (10 ng/mL) stimulated cells. Fold change was relative to untreated controls. n = 5. b, Representative immunoblots of synthetic marker proteins in BCKA-stimulated cells. n = 6. c, Confocal microscopy images and quantitation represent COL4 synthesis and deposition in BCKA-treated PASMCs. Fold change was relative to control cells. n = 5. d,e, Protein ( d ) and mRNA ( e ) expression of synthetic marker genes in PASMCs transfected with control siRNA (siCtrl) or human BCAT1 and BCKDHA1 siRNAs (siBCAT1+siBCKDHA1) with or without BCKA treatment. Fold change was calculated relative to siCtrl-transfected and untreated cells. n = 3 ( d ) and 6 ( e ). f,g, Protein ( f ) and mRNA ( g ) expression of synthetic marker genes in PASMCs transfected with siCtrl or human BCAT2 and BCKDK siRNAs (siBCAT2+siBCKDK) with or without BCKA treatment. Fold change was calculated relative to siCtrl-transfected and untreated cells. n = 3 ( f ) and 12 ( g ). h, COL1A1 and VIM protein expression in control and HIF1 α knockdown PASMCs in the presence or absence of BCKAs. n = 8. i, Confocal microscopy and quantitation showing COL1A1 synthesis and deposition in control and HIF1α knockdown PASMCs in the presence or absence of BCKAs. Fold change was relative to control cells. n = 6. j, Collagen gel contraction assay illustrating the relative contracted area of collagen gels after 8-hour of detachment in control and HIF1α knockdown PASMCs with or without BCKAs. n = 6. All data are presented as mean ± SD. One-way ANOVA followed by Dunnett’s ( a ) or Tukey’s post-hoc test ( e , g , i , j ), or Kruskal-Wallis test followed by Dunn’s test (COL4A1 in g ), or Mann-Whitney U test ( c ) was applied when compared to untreated controls ( a , c ), siCtrl-transfected and untreated or BCKA-treated PASMCs ( e , g , i , j ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Expressing, Marker, Western Blot, Confocal Microscopy, Quantitation Assay, Control, Transfection, Knockdown, Collagen Gel Contraction Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a,b, mRNA ( a ) and protein ( b ) expression of key BCKA metabolic genes in human lung tissues of idiopathic PAH (IPAH) patients and transplantation-failed donors (Ctrl). Fold change was calculated relative to control donors. n = 10 individuals ( a ) and 8 individuals ( b ). c, The levels of BCKAs and BCAAs in the lungs of IPAH patients and control donors. Fold change was calculated relative to control donors. n = 7 individuals. d, Immunofluorescent images and quantitation of HIF1α protein in lung tissues of IPAH patients and control donors. Fold change was calculated relative to control donors. n = 5 individuals. e-g, Protein levels of four BCKA metabolic enzymes in lung tissues of control and PAH rats induced by MCT ( e ), hypoxia (10% O 2 , f ), or Sugen5416 and hypoxia treatments ( g ). n = 4 ( e , g ) and 3-4 ( f ) rats per condition. h, Immunofluorescence images and quantitation of HIF1α protein in control and MCT-treated rat lung tissues. n = 4 rats. All data are presented as mean ± SD. Student’s t test ( a , c , d ) or Mann-Whitney U test ( h ) was conducted when compared to lungs from failed donors ( a , c , d ) or control rats ( h ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Expressing, Transplantation Assay, Control, Quantitation Assay, Immunofluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells

doi: 10.1101/2024.05.29.595538

Figure Lengend Snippet: a, mRNA expression of four BCKA metabolic enzymes, glycolytic gene PFKFB3 , and synthetic markers in PASMCs from IPAH patients and commercially available control donors. Fold change was relative to control donors. n = 5 individuals. b, Protein levels of four BCKA metabolic enzymes and COL4 in PASMCs from IPAH and commercially available control donors. Fold change was relative to control donors. n = 4-5 individuals. c, Volcano plot from LC-MS metabolomic profiling showing the levels of 139 metabolites in normal and IPAH PASMCs. n = 5 individuals. d, LC-MS measurements of KIC/KMV, BCAAs, and glycolytic metabolites in normal and IPAH PASMCs. Fold change was calculated relative to normal PASMCs. n = 5 individuals. e,f, mRNA expression of HIF1α transcriptional target genes in glucose metabolism ( e ) and protein expression of HIF1α, PFKFB3, and COL1A1 ( f ) in BCKA-treated IPAH-PASMCs. Fold change was calculated relative to untreated IPAH-PASMCs. n = 7 ( e ) and 8 ( f ) from 4 individuals. g, Lactate secretion by IPAH-PASMCs treated with or without BCKAs. Fold change was calculated relative to untreated IPAH-PASMCs. n = 8 from 4 individuals. h, Confocal microscopy and quantitation results showing collagen synthesis and deposition in IPAH-PASMCs treated with vehicle control or BCKAs. Fold change was calculated relative to untreated IPAH-PASMCs. n = 5 from 4 individuals. All data are presented as mean ± SD. Student’s t test or Mann-Whitney U test was used when compared to PASMCs from normal control donors ( a , b , d ) or untreated PASMCs from IPAH patients ( e-h ).

Article Snippet: For immunocytochemistry assay, cells were seeded on 4-well Nunc Lab-Tek chamber slide and fixed with 4% paraformaldehyde for 10 min. Tissue and cell slides were then blocked with 1% BSA and 10% normal goat serum in PBS at room temperature for 1 hour followed by overnight incubation at 4°C with primary antibodies (1:50 dilution) against human HIF1α (cat # 610958, BD Biosciences), rat HIF1α (cat # NB100-105, Novus Biologicals), human smooth muscle actin alpha (αSMA; cat # ab124964, Abcam), rat αSMA (cat # 5694, Abcam), human COL1A1 (cat # NBP1-30054, Novus Biologicals), or human COL4 (cat # NB120-6586, Novus Biologicals), and then Alexa Fluor ® 568 goat anti-mouse (cat # ab175473, Abcam), 568 goat anti-rabbit (cat # 175471, Abcam), 488 goat anti-mouse (cat # 1500113, Abcam), or 488 goat anti-rabbit (cat # ab150077, Abcam) IgG secondary antibody (1:200 dilution) at room temperature for 1 hour.

Techniques: Expressing, Control, Liquid Chromatography with Mass Spectroscopy, Confocal Microscopy, Quantitation Assay, MANN-WHITNEY