antibody against pdk2  (Servicebio Inc)

Journal: International Journal of Molecular Sciences

Article Title: Targeting Lactate Dehydrogenase A with Catechin Resensitizes SNU620/5FU Gastric Cancer Cells to 5-Fluorouracil

doi: 10.3390/ijms22105406

Figure Lengend Snippet: Primer sequences used for qRT–PCR.

Article Snippet: Further, antibodies against PDK2 and PDK4 were purchased from Signalway Antibody (Signalway Antibody, Dallas, TX, USA), those against PDK1 was obtained from Enzo Life Sciences (Enzo Life Sciences, Farmingdale, NY, USA), and those against B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein were purchased from Novus Biologicals (Novus Biologicals, Littleton, CO, USA).

Techniques:

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: The expression of Pdk isoform (Pdk1-4) mRNAs in the hypothalamus at 1, 2, 3, and 6 w following STZ injection ( a ) was assessed by real-time RT-PCR. mRNA expression profiles are displayed as the fold increase of gene expression normalized to Gapdh (* p = 0.0463, 1 w; * p = 0.0249, 2 w; ** p = 0.0063, 3 w; and * p = 0.0212, 6 w for Pdk2 ). PDK2 (* p = 0.0442, STZ), phosphorylated-PDH (p-S 293 -PDH: ** p = 0.0048, STZ; and p-S 300 -PDH: *** p = 0.0002, STZ), and PDH-E1 protein levels in the hypothalamus at 3 w post-STZ injection ( b ) were assessed by Western blot analysis. Western blot band quantification for p-PDH was based on normalization to PDH-E1, and PDK2 and PDH-E1 were normalized to α-tubulin. Immunofluorescence analyses show the expression of PDK2 in GFAP-positive astrocytes ( c ), βIII-tubulin-positive neurons ( d ), and Iba-1-positive microglia ( e ) in the hypothalamus at 3 w post-STZ injection. Inserts show the representative double-labeled cells. Quantification of the percentage of PDK2-positive or -negative astrocytes (### p = 1.5E-12, not co-localized with PDK2; and ### p = 1.6E-12, co-localized with PDK2), neurons (# p = 0.0119, not co-localized with PDK2; and # p = 0.0143, co-localized with PDK2), or microglia. Pearson’s correlation coefficients for co-localization per mm 2 (*** p = 1.02E-5, STZ for c ; and *** p = 5.2E-5, STZ for d ) are shown in the adjacent graphs. Results were obtained from three animals for each condition. Microscopic data were evaluated in five randomly selected fields captured at the same magnification. Scale bars indicate 200 µm. * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals. One-way ANOVA ( a ), two-way ANOVA ( c – e , left) with Tukey’s post hoc test; Student’s t -test ( b – e , right), n = 3 ( a , b ), and n = 5 ( c – e ); mean ± SEM. Value of “n” indicates the number of animals. Source data are provided as a Source data file. w week(s); STZ streptozotocin; ND not detected; ns not significant.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Expressing, Injection, Quantitative RT-PCR, Western Blot, Immunofluorescence, Labeling

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: GFAP (# p = 0.0101, KO + STZ for hypothalamus; ###p = 0.0003, KO + STZ for ARC; and # p = 0.0192, KO + STZ for PVN) and Iba-1 (# p = 0.0170, KO + STZ for hypothalamus, ## p = 0.0090, KO + STZ for ARC; and # p = 0.0277, KO + STZ for PVN) immunofluorescence staining and image analysis were performed using WT and Pdk2 -deficient mice at 3 w post-STZ injection ( a , b ). Microscope data were gathered using five to six randomly selected fields captured at the same magnification. Scale bars indicate 400 µm ( a , b , hypothalamus) and 200 µm ( a , b , ARC and PVN). The relative expression of Tnf-α (#p = 0.0112, KO + STZ), Il-1β (###p = 0.0003, KO + STZ), and Il-6 (##p = 0.0016, KO + STZ) mRNAs in the hypothalamus after 3 w of STZ injection ( c ) was evaluated by real-time RT-PCR. Results for mRNA expression are displayed as the fold increase in gene expression normalized to Gapdh . Lactate concentration in hypothalamic tissues collected from WT and Pdk2 KO mice at 3 w post-STZ/vehicle injection was measured with an HPLC analyzer ( d ). The quantification of lactate concentration (### p = 0.0002, KO + STZ) from HPLC graphs is shown. Food intake (* p = 0.0017, KO + STZ at 22 d) ( e ) and fasting blood (* p = 0.0033, KO + STZ at 23 d) ( f ) and CSF glucose (# p = 0.0101, KO + STZ) ( g ) levels in mice were assessed following STZ injection at the indicated time points. Fasting blood glucose levels were measured in the pair-feeding condition following STZ administration ( h ). The pair-feeding condition was established based on the consumption of food by diabetic KO animals ( p = 0.9997, KO + STZ for PF at 3 w). The relative expression of Npy (## p = 0.0031, KO + STZ), Agrp (### p = 0.0004, KO + STZ), and Pomc (# p = 0.0158, KO + STZ) mRNA in the hypothalamus after 3 w of STZ injection ( i ) was evaluated by real-time RT-PCR. mRNA expression results are displayed as the fold increase of gene expression normalized to Gapdh . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the WT/KO control animals ( a – d , g – i ) or STZ-injected animals ( e , f ); # p < 0.05 or ## p < 0.01 versus indicated groups. Two-way ANOVA with Tukey’s post hoc test, n = 6 for KO + Control and 5 for other groups ( a , b ), n = 5 for control and 6 for STZ groups ( d ), n = 6 ( e – h ), and n = 3 ( c , i ); mean ± SEM. Value of “ n ” indicates the number of animals. Source data are provided as a Source data file. w week(s); d day(s); WT wild-type; KO knock out; STZ streptozotocin; ARC arcuate nucleus; PVN paraventricular nucleus; IR immunoreactivity; RF regular feeding; PF pair feeding; ns not significant.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Immunofluorescence, Staining, Injection, Microscopy, Expressing, Quantitative RT-PCR, Concentration Assay, Knock-Out

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: The relative expression of Pdk2 (*** p = 7.6E-6, Gluc 16 mM; and *** p = 3.6E-7, Gluc 25 mM) ( a ), Tnf-α (*** p = 0.0001, Gluc 16 mM; and *** p = 8.6E-6 Gluc 25 mM), Il-1β (*** p = 2.2e-5, Gluc 16 mM; and *** p = 2.7E-9, Gluc 25 mM), and Il-6 (*** p = 1.8E-5, Gluc 16 mM; and *** p = 7.5E-12, Gluc 25 mM) mRNA in cultured astrocytes was assessed after high glucose (16 mM and 25 mM) treatment for 24 h ( c ). Similarly, the expression of Pdk 2 mRNA (### p = 1.7E-5, Gluc 25 mM + GSK) in astrocytes was assessed after co-treatment with high glucose (25 mM) and GSK0660 (a PPARβ/δ antagonist, 1 µM) for 24 h ( b ). The expression of Tnf-α (## p = 0.0022, KO + Gluc 25 mM), Il-1β (### p = 2.3E-9, KO + Gluc 25 mM) and Il-6 (### p = 0.0002, KO + Gluc 25 mM) mRNA was examined in cultured astrocytes isolated from WT and Pdk2 KO mice ( d ). Extracellular lactate was assessed after primary astrocytes isolated from WT and Pdk2 KO mice were exposed to high glucose for 24 hr by HPLC analysis ( e ). ECAR (* p = 0.0331, KO + Gluc 25 mM at 60 min; and ###p = 7.2E-6, KO + Gluc 25 mM at glycolytic capacity) ( f – g ) and OCR (* p = 0.0289, KO + Gluc 25 mM at 60 min) ( h ) were measured after primary astrocytes isolated from WT and Pdk2 KO mice were treated with high glucose (25 mM) for 24 h by an XF 24 Seahorse analyzer. Relative expression of Tnf-α (# p = 0.0340, Gluc 25 mM+Oxamate; and # p = 0.0246, Gluc 25 mM+GSK2837808A), Il-1β (## p = 0.0080, Gluc 25 mM + Oxamate; and ##p = 0.0020, Gluc 25 mM + GSK2837808A) , Il-6 (###p = 2.1E-5, Gluc 25 mM+Oxamate; and ## p = 0.0012, Gluc 25 mM + GSK2837808A) mRNA in astrocytes was assessed after co-treatment with high glucose (25 mM) and Oxamate (5 mM) or GSK2837808A (2 µM) for 24 h ( i ). The schematic diagram represents the timeline and condition for the hypothalamic slice culture experiment ( j ). Levels of TNF-α protein (## p = 0.0099, AAV5-GFAP-mCherry-Cre + Gluc 25 mM) in the culture media ( k ) and expression of Tnf-α (#p = 0.0294, AAV5-GFAP-mCherry-Cre+Gluc 25 mM) and Il-1β (## p = 0.0058, AAV5-GFAP-mCherry-Cre + Gluc 25 mM) mRNAs ( l ) in the cultured hypothalamus isolated from AAV5-GFAP-eYFP or AAV5-GFAP-mCherry-Cre-injected mice were assessed after high glucose treatment. Protein level was measured by ELISA. mRNA expression was assessed by real-time RT-PCR, and results are displayed as the fold increase of gene expression normalized to Gapdh . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the non-treated group; # p < 0.05, ## p < 0.01, or ### p < 0.001 versus indicated groups. One-way ANOVA ( a – c , i , k , l ) and two-way ANOVA with Tukey’s post hoc test ( d – h ), n = 6 ( a , c , d ), n = 3 ( b , i , k , l ), and n = 3–4 ( e – h ); mean ± SEM. Value of “ n ” indicates the number of sister wells in culture plates. Source data are provided as a Source data file. A diagram showing the hypothetical involvement of the PDK2-lactic acid axis in high glucose-induced inflammatory activation of astrocytes ( m ). Gluc glucose; GSK GSK0660.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Expressing, Cell Culture, Isolation, Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Activation Assay

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: To determine the role of hypothalamic PDK2 or lactic acid in the regulation of food intake, we administered AZD7545 (6.4 nM of CSF concentration), GSK2837808A (2 μM of CSF concentration), or oxamate (25 μg) by multiple icv injections into mice starting from 5 d post-STZ injection to the following indicated time points. The schematic diagram represents the timeline of experimentation and route of administration of the inhibitors ( a ). The relative expression of Tnf-α , Il-1β , and Il-6 mRNA in the hypothalamic tissues isolated from AZD7545 or GSK2837808A or oxamate and STZ-injected mice was evaluated by real-time RT-PCR ( b ). Food intake ( c , d ) was assessed following AZD7545, GSK2837808A, or oxamate and STZ administration. Results for mRNA expression are displayed as the fold increase of mRNA expression normalized to Gapdh . * p or § p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals ( b ) or STZ + vehicle-treated animals ( c , d ); # p < 0.05, ## p < 0.01, or ### p < 0.001 versus indicated groups. One-way ANOVA ( b ) and two-way ANOVA with Tukey’s post hoc test ( c , d ), n = 4 ( b ), and n = 9 for STZ and 5 for other groups ( c , d ); mean ± SEM. Value of ‘n’ indicates the number of animals. Source data are provided as a Source data file. d day(s); STZ streptozotocin.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Concentration Assay, Injection, Expressing, Isolation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: To further determine the role of hypothalamic PDK2 in diabetes-induced neuroinflammation and dysregulation of feeding behavior, we administered Ad-PDK2-GFP or Ad-GFP (1.0 × 10 9 pfu) via the icv route 1 w before STZ injection. The schematic diagram shows the experimentation timeline and the route of administration ( a ). PDK2 protein levels (### p = 4.5E-5, KO + Ad-PDK2-GFP + STZ) in the hypothalamus after 3 w of STZ and 4 w of adenovirus injection were assessed by Western blot analysis ( b ). Western blot band quantification for PDK2 protein levels normalized to α-tubulin. The relative expression of Tnf-α (## p = 0.0030, KO + Ad-PDK2-GFP + STZ), Il-1β (# p = 0.0380, KO + Ad-PDK2-GFP + STZ), and Il-6 (## p = 0.0020, KO + Ad-PDK2-GFP + STZ) mRNA in the hypothalamic tissues isolated from Ad-PDK2-GFP or Ad-GFP and WT mice was evaluated by real-time RT-PCR ( c ). Food intake (* p = 0.0130, KO + Ad-PDK2-GFP + STZ at 28 d) ( d ) and fasting blood glucose levels (* p = 0.0127, KO + Ad-PDK2-GFP + STZ at 28 d) ( e ) were assessed following Ad-PDK2-GFP or Ad-GFP and STZ administration. Arrows indicate the time points of adenovirus (Ad) and STZ administration. The relative expression of Npy (# p = 0.0489, KO + Ad-PDK2-GFP + STZ), Agrp (## p = 0.0090, KO + Ad-PDK2-GFP + STZ), and Pomc (#p = 0.0270, KO + Ad-PDK2-GFP + STZ) mRNA in the hypothalamic tissues isolated from Ad-PDK2-GFP or Ad-GFP and STZ-injected mice just after completion of behavioral assessments was evaluated by real-time RT-PCR ( f ). Results for mRNA expression are displayed as the fold increase of mRNA expression normalized to Gapdh . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals ( b , c , f ) or STZ/Ad-GFP + STZ-treated animals ( d , e ); # p < 0.05, ## p < 0.01 between the indicated groups. One-way ANOVA ( b , c , f ) and two-way ANOVA with Tukey’s post hoc test ( d , e ), n = 3 (b), n = 4 ( c , f ), and n = 6 ( d , e ); mean ± SEM. Value of “ n ” indicates the number of animals. Source data are provided as a Source data file. d day(s); STZ streptozotocin; pfu plaque-forming unit; ns not significant; ND not detected.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Injection, Western Blot, Expressing, Isolation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: p-AMPK (# p = 0.0157, KO + STZ) and AMPK protein levels in the hypothalamic tissues isolated from WT and Pdk2 KO mice at 3 w post-STZ/vehicle injection were assessed by Western blot analysis ( a ). Co-immunofluorescence staining of mouse brain tissues showed p-AMPK and βIII-tubulin-positive neurons (### p = 1.4E-7, not co-localized with p-AMPK; and (### p = 3.7E-7, co-localized with p-AMPK) in the ARC at 3 w post-STZ/vehicle injection ( b ). The percentage of p-AMPK-positive neurons was calculated from the total number of cells present per mm 2 using six randomly selected fields. Scale bar indicates 100 µm. The expression of p-AMPK (** p = 0.0051, lactic acid) and AMPK proteins ( c ), as well as Agrp ( d ) in mHypoE-N41 cells, and Pomc mRNA in mHypoE-N43/5 cells ( e ) after treating with lactic acid (7.1 mM) for 72 h was assessed by Western blot and real-time RT-PCR, respectively. The effect of Compound C (16 µM) and metformin (100 µM) on lactic acid-induced increased expression of Agrp mRNA (## p = 0.0011, lactic acid+ComC) in mHypoE-N41 cells was observed ( d ). Results for mRNA expression are displayed as the fold increase of gene expression normalized to Gapdh . The diagram shows the proposed lactic acid-AMPK axis in the regulation of the AgRP ( f ). Representative traces of I NMDA induced by NMDA (100 μM) in the absence or presence of l -lactate (7.1 mM) in NPY/AgRP-expressing neurons (left) and POMC-expressing neurons (right) ( g ). l -lactate-induced changes of the NMDA-induced currents in NPY and POMC neurons (## p = 0.0006, POMC) ( h ). Representative traces of sEPSC NMDA in the absence (left) or presence (right) of l -lactate in NPY neurons ( i ). Cumulative probability distribution for inter-event interval (left) and amplitude (right) of sEPSC NMDA with or without l -lactate ( j ). The plot includes 195 events for control (thin lines) and 143 events for l -lactate (thick lines). Representative traces before, during, and after the application of NMDA in the absence (left) or presence (right) of l -lactate in NPY neurons in the current-clamp condition ( k ). NMDA-induced changes of action potential frequency in the absence or presence of l -lactate in NPY neurons (* p = 0.0220, NMDA + l -lactate) ( l ). Open and closed circles represent the value from individual neurons and their average respectively. Insets represent the l -lactate-induced changes in the frequency (left) and amplitude (right) of sEPSC NMDA . Dotted lines represent the relative control of basal frequency and amplitude of sEPSC NMDA . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals/cells. Two-way ANOVA ( a , b ), one-way ANOVA with Tukey’s post hoc test ( d , h ), two-tailed Student’s t -test ( c , e ), two-tailed paired t -test ( h , j , l ), unpaired t -tes t , n = 9 individual neurons ( h ), n = 195 events for control and 143 for l -lactate ( j ), n = 6 individual neurons ( l ) n = 3 animals per group ( a ) or sister wells ( c – e ), and n = 5 animals per group ( b ); mean ± SEM. Source data are provided as a Source data file. ComC Compound C; Met metformin; L-lac l -lactate; Amp Amplitude; Freq Frequency; NC negative control; ns not significant.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Isolation, Injection, Western Blot, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test, Negative Control

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: To determine the role of hypothalamic PDK2 in the regulation of neuroinflammation and feeding behavior, we administered AAV5-GFAP-mCherry-Cre or AAV5-GFAP-eYFP (4.3 × 10 9 virus particles) into the mediobasal hypothalamus of Pdk2 floxed mice, bilaterally, using a stereotaxic device 1 w before STZ injection. The schematic diagram presents the experiment timeline and route of administration ( a ). The relative expression of Tnf-α (# p = 0.0141, AAV5-GFAP-mCherry-Cre+STZ), IL-1β (# p = 0.0316, AAV5-GFAP-mCherry-Cre+STZ), and IL-6 (## p = 0.0016, AAV5-GFAP-mCherry-Cre+STZ) mRNA in the hypothalamic tissues isolated from AAV5-GFAP-mCherry-Cre or AAV5-GFAP-eYFP and STZ-injected mice was evaluated by real-time RT-PCR ( b ). GFAP (## p = 0.0020, AAV5-GFAP-mCherry-Cre + STZ) and Iba-1 (## p = 0.0053, AAV5-GFAP-mCherry-Cre+STZ) immunofluorescence staining revealed their increased immunoreactivity in the hypothalamus of diabetes Pdk2 floxed mice, but Pdk2 -deficiency in hypothalamic astrocytes attenuated such an increase in the immunoreactivities at 3 w post-STZ injection ( c ). Microscope data were gathered using five randomly selected fields captured at the same magnification. Scale bar indicates 200 µm. Lactate concentration (## p = 0.0030, AAV5-GFAP-mCherry-Cre+STZ) in hypothalamic tissues collected from AAV5-GFAP-mCherry-Cre- or AAV5-GFAP-eYFP- and STZ-injected mice was measured with an HPLC analyzer ( d ). Quantification of the lactate concentration from HPLC graphs is shown. Food intake (* p = 0.0393, AAV5-GFAP-mCherry-Cre+STZ at 4 w) was assessed following AAV5-GFAP-mCherry-Cre or AAV5-GFAP-eYFP and STZ administration ( e ). Arrows indicate the time points of adeno-associated virus (AAV), and STZ administration. The relative expression of Npy (# p = 0.0140, AAV5-GFAP-mCherry-Cre+STZ), Agrp (# p = 0.0380, AAV5-GFAP-mCherry-Cre+STZ) and Pomc (## p = 0.0090, AAV5-GFAP-mCherry-Cre + STZ) mRNA in hypothalamic tissues isolated from AAV5-GFAP-mCherry-Cre- or AAV5-GFAP-eYFP- and STZ-injected mice just after completion of behavioral assessments was evaluated by real-time RT-PCR ( f ). mRNA expression results are displayed as the fold increase of mRNA expression normalized to Gapdh . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals ( b – d , f ) or STZ/AAV5-GFAP-eYFP+STZ-treated animals ( e ); # p < 0.05, ## p < 0.01, or ### p < 0.001 versus indicated groups. One-way ANOVA ( b – d , f ) and two-way ANOVA ( e ) with Tukey’s post hoc test, n = 3 ( b , d , f ), n = 5 ( c ), and n = 6 ( e ); mean ± SEM. Value of “ n ” indicates the number of animals. Source data are provided as a Source data file. w week(s); IR immunoreactivity.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Injection, Expressing, Isolation, Quantitative RT-PCR, Immunofluorescence, Staining, Microscopy, Concentration Assay

Journal: Oncotarget

Article Title: Dichloroacetate potentiates tamoxifen-induced cell death in breast cancer cells via downregulation of the epidermal growth factor receptor

doi: 10.18632/oncotarget.10999

Figure Lengend Snippet: ( A and C ) MCF7 cells were treated with siRNA against PDK (A) or 20–50 μM DCA (C) for 36 h, and the cell lysates were subjected to Western blotting. The blot is representative of two independent experiments. B. MCF7 cells were treated with siRNA against PDK1, PDK2, PDK3, or PDK4 for 48 h, and the cell lysates were subjected to Western blotting. ( D and E ) MCF7 cells were treated with or without 10 μM tamoxifen and/or 20 mM DCA for 48–72 h. The cell morphological changes (D) were observed under an inverted microscope, and the images are representative of three independent experiments. Cell viability (E) was assessed using an MTT assay. Data are presented as the mean of triplicate samples, and error bars reflect the SD. ( F ) MCF7 cells were treated with or without 10 μM tamoxifen or/and 20 mM DCA for 24 and 48 h. Cell death was evaluated by flow cytometry after Annexin V and PI staining. Data are presented as the mean of triplicate samples, and error bars reflect the SD. * p < 0.05;*** p < 0.001 compared to untreated. ns, nonsignificant.

Article Snippet: Antibodies against PDK2 and PDK3 were acquired from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Western Blot, Inverted Microscopy, MTT Assay, Flow Cytometry, Staining