Journal: Aging Cell

Article Title: Silencing of the Metabolic Gene HKDC1 Is Associated With Aging and Neurodegeneration in Mice and Humans

doi: 10.1111/acel.70419

Figure Lengend Snippet: Loss of HKDC1 in the brain is associated with inflammation and senescence. (A) Schematic representation of mouse genotypes (HKDC1 fl/fl and BKO) and time‐points of analysis of mitochondrial health and neuroinflammation markers. (B) RNA expression of inflammatory markers in 9‐month‐old HKDC1 fl/fl and BKO male mice ( n = 6) using 18S as an internal control. Values are mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by student t ‐test (C) Immunoblot of COX‐2 expression in 9‐month old HKDC1 fl/fl and BKO male and female mice ( n = 2). Actin was used as a loading control. (D) Light microscopy images of HKDC1fl/fl and BKO brain sections at 40× magnification. Scale bars, 25 μm. n = 7–9 cells/group from experiments repeated three times. (E) RNA expression of senescence markers in 9‐month‐old HKDC1 fl/fl and BKO male mice ( n = 6) using 18S as an internal control. (F, G) Protein and RNA expression of mitochondrial health marker PINK1 in 9‐month‐old HKDC1 fl/fl and BKO male mice ( n = 6) using Actin (F) and 18S (G) as an internal control. (H) Bar graph showing relative concentration of Aβ‐42 in the serum of 9‐month‐old HKDC1 fl/fl and BKO mice. APP‐PS1 Alzheimer's disease mouse model (9‐month) was used as a positive control. (I) Western blot of p‐tau expression in 9‐month‐old HKDC1 fl/fl and BKO male mice ( n = 4). 5X‐ FAD Alzheimer's disease mouse model (6‐month) was used as a positive control. Values are mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by student t ‐test (A, C) with Welch's correction or ordinary one‐way ANOVA (H). Effect size ( r ) = −0.63 (d) = −1.63 (TNFα), ( r ) = −0.75 (d) = −2.27 (COX‐2), ( r ) = −0.957 (d) = −6.63 (IL‐1β) (B), ( r ) = −0.68 (d) = −1.85 (p21), ( r ) = −0.991 (d) = −15.23 (p53) (E), ( r ) = 0.87 (d) = 3.63 (G).

Article Snippet: Membranes were blocked with 5% nonfat dried milk in tris‐buffered saline supplemented with 0.1% Tween‐20 for 1 h at room temperature, followed by incubation with desired primary antibodies overnight at 4°C [β‐actin (Cell Signaling technologies, 13E5), GAPDH (Abcam, ab8245), HKDC1 (Abcam, ab228729), HK1 (Abcam, ab154839), COX‐2 (Proteintech, #66351–1‐Ig), Cox‐IV (Cell Signaling technologies, #4850 T), Phospho‐tau (Invitrogen, #MN10120), TFEB (Cell Signaling technologies, #37785S), LC3B (Cell Signaling technologies, #2775), Pink1 (Proteintech, #23274–1‐AP), Parkin1 (Cell Signaling technologies, #4211 T), p‐Parkin1 (Abcam, #ab315376), and P62 (Cell Signaling technologies, #5114 T)].

Techniques: RNA Expression, Control, Western Blot, Expressing, Light Microscopy, Marker, Concentration Assay, Positive Control

Journal: Aging Cell

Article Title: Silencing of the Metabolic Gene HKDC1 Is Associated With Aging and Neurodegeneration in Mice and Humans

doi: 10.1111/acel.70419

Figure Lengend Snippet: Loss of HKDC1 expression in CCF‐STTG1 human astrocyte cell line causes inflammation, increase in reactive oxygen species (ROS) production and autophagy. (A) Schematic representation of CCF‐STTG1 astrocyte cells indicating silencing of HKDC1 expression, RNA and protein extraction. (B) Immunoblot of HKDC1 expression in CCF‐STTG1 astrocytes to confirm siRNA mediated silencing of HKDC1. (C) RT‐PCR of HKDC1 in siScr and siHKDC1 CCF‐STTG1 astrocytes using 18S as an internal control ( n = 4). (D–G) RNA expression of mitophagy marker PINK1 (D), inflammatory markers IL‐6 (E), S100B (F), IL‐1β (G), and TNF‐α (H) using 18S as an internal control ( n = 4). (I) Schematic representation of CCF‐STTG1 silencing with FCCP treatment for measurement of ROS production and autophagy. (J) DCF fluorescence results from siScr and siHKDC1 astrocytes following 4 h treatment with vehicle (DMSO) and 5 μM, 10 μM, and 20 μM FCCP, as compared to 100 μM treatment with TBHP for maximal ROS production. Values are mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by student t ‐test (C–G) with Welch's correction or two‐way ANOVA (J).

Article Snippet: Membranes were blocked with 5% nonfat dried milk in tris‐buffered saline supplemented with 0.1% Tween‐20 for 1 h at room temperature, followed by incubation with desired primary antibodies overnight at 4°C [β‐actin (Cell Signaling technologies, 13E5), GAPDH (Abcam, ab8245), HKDC1 (Abcam, ab228729), HK1 (Abcam, ab154839), COX‐2 (Proteintech, #66351–1‐Ig), Cox‐IV (Cell Signaling technologies, #4850 T), Phospho‐tau (Invitrogen, #MN10120), TFEB (Cell Signaling technologies, #37785S), LC3B (Cell Signaling technologies, #2775), Pink1 (Proteintech, #23274–1‐AP), Parkin1 (Cell Signaling technologies, #4211 T), p‐Parkin1 (Abcam, #ab315376), and P62 (Cell Signaling technologies, #5114 T)].

Techniques: Expressing, Protein Extraction, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, RNA Expression, Marker, Fluorescence

Journal: Cancers

Article Title: Prognostic and Predictive Implications of PTEN in Breast Cancer: Unfulfilled Promises but Intriguing Perspectives

doi: 10.3390/cancers11091401

Figure Lengend Snippet: Phase II and phase III clinical trials with translational analyses exploring the predictive role of PTEN in HER2-positive breast cancer.

Article Snippet: , Trastuzumab + Lapatinib , NCT00206427 [ ] , II , EBC , Neoadjuvant , CT + Trastuzumab + Lapatinib , 49 , IHC clone AF847, R and D Systems (protein level: 0 versus 1+ versus 2+ versus 3+) , pCR rates in low PTEN versus normal-PTEN: 92.3% versus 41.2%.

Techniques: Clinical Proteomics, Immunofluorescence, Adjuvant, Staining, Modification, Membrane, Expressing

Journal: Science Advances

Article Title: CD38 reduces mitochondrial fitness and cytotoxic T cell response against viral infection in lupus patients by suppressing mitophagy

doi: 10.1126/sciadv.abo4271

Figure Lengend Snippet: ( A ) Percentage of depolarized mitochondria in control, CD38-overexpressing CD8 + T cells, or CD38-overexpressing cells treated with SRT1720, or one of the CD38 inhibitors (78c or MK-0159). ( B to D ) Percentage of positive cells expressing CD107a (B), granzyme B (C), and IFN-γ (D) in control, CD38-overexpressing CD8 + T cells, or CD38-overexpressing cells treated with SRT1720, or one of the CD38 inhibitors (78c or MK-0159). ( E ) MFI of LC3B staining in control or CD38-overexpressed CD8 + T cells. ( F ) Counts of LC3B-positive dots per cell of control or CD38-overexpressed CD8 + T cells. ( G ) MFI of PINK1 in control, CD38-overexpressing CD8 + T cells, or CD38-overexpressing cells treated with SRT1720. ( H ) Percentage of Parkin positive of the TOMM20-positive area in control, CD38-overexpressing CD8 + T cells, or CD38-overexpressing cells treated with SRT1720. ( I ) MFI of PINK1 in CD38 hi CD8 + and CD38 lo CD8 + T cells from lupus patient peripheral blood. ( J ) Percentage of Parkin positive of the TOMM20-positive area in CD38 hi CD8 + and CD38 lo CD8 + T cells from lupus patient peripheral blood. Data are means ± SD; statistical analysis by two-tailed t test (A to H and J), paired t test (I), ns = P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For flow cytometry, CD8 + T cells from human peripheral blood were stained with the following antibodies or reagents, listed with clones, vendor, and catalog numbers: anti-human CD4 (clone RPA-T8, 300521), anti-human CD8a (clone SK1, BioLegend, 344718), anti-human CD8a (clone HIT8a, BioLegend, 300908), anti-human CD38 (clone HIT2, BioLegend, 303516 and 303510), allophycocyanin (APC) mouse immunoglobulin G1 (IgG1), κ isotype control (BD, 555751), anti–green fluorescent protein (GFP) antibody (Rockland, 600-102-215), anti–IFN-γ (clone 4S.B3, BioLegend, 502509), anti–granzyme B (clone GB11, BD, 560212), anti-CD107a (clone H4A, BioLegend, 328608), PINK1 antibody (Rockland, 600-401-GU5), anti-Rab4 antibody (EPR3043, Abcam, ab109009), goat anti-rabbit IgG secondary antibody, and APC (Invitrogen, A-10931).

Techniques: Control, Expressing, Staining, Two Tailed Test

Journal: Science Advances

Article Title: CD38 reduces mitochondrial fitness and cytotoxic T cell response against viral infection in lupus patients by suppressing mitophagy

doi: 10.1126/sciadv.abo4271

Figure Lengend Snippet: ( A and B ) Percentage of positive cells expressing CD107a (A) and granzyme B (B) in gp33 + CD8 + T cells 8 days after LCMV Armstrong infection from diseased BXD2 treated with control water or MK-0159. ( C ) Percentage of depolarized mitochondria in gp33 + CD8 + T cells 8 days after LCMV Armstrong infection from diseased BXD2 treated with control water or MK-0159. ( D ) LCMV viral load 8 days after LCMV Armstrong infection using qPCR of glycoprotein gene normalized by tissue ACTB in diseased BXD2 treated with control water or MK-0159. ( E to G ) Representative liver histology 8 days after LCMV Armstrong infection from diseased BXD2 treated with control water (E) or MK-0159 (F) and their respective hepatitis activity index (G). ( H and I ) Percentage of positive cells expressing CD107a (H) and granzyme B (I) in gp33 + CD8 + T cells 8 days after LCMV Armstrong infection from diseased BXD2 treated with control NP or NP-MK-0159. ( J to M ) Representative liver histology 8 days after LCMV Armstrong infection from diseased BXD2 treated with NP-control with the presentation of necrosis (J) or portal and periportal inflammation (K) or treated with NP-MK-0159 (L) and their respective hepatitis activity index (M). ( N ) LCMV viral load 8 days after LCMV Armstrong infection using qPCR of glycoprotein gene normalized by tissue ACTB in diseased BXD2 treated with NP-control or NP-MK-0159. ( O ) Percentage of depolarized mitochondria in gp33 + CD8 + T cells 8 days after LCMV Armstrong infection from diseased BXD2 treated with NP-control or NP-MK-0159. ( P and Q ) MFI of PINK1 (P) and LysoSensor Green (Q) in gp33 + CD8 + T cells 8 days after LCMV Armstrong infection from diseased BXD2 treated with NP-control or NP-MK-0159. Data are means ± SD; statistical analysis by two-tailed t test. * P < 0.05, ** P < 0.01.

Article Snippet: For flow cytometry, CD8 + T cells from human peripheral blood were stained with the following antibodies or reagents, listed with clones, vendor, and catalog numbers: anti-human CD4 (clone RPA-T8, 300521), anti-human CD8a (clone SK1, BioLegend, 344718), anti-human CD8a (clone HIT8a, BioLegend, 300908), anti-human CD38 (clone HIT2, BioLegend, 303516 and 303510), allophycocyanin (APC) mouse immunoglobulin G1 (IgG1), κ isotype control (BD, 555751), anti–green fluorescent protein (GFP) antibody (Rockland, 600-102-215), anti–IFN-γ (clone 4S.B3, BioLegend, 502509), anti–granzyme B (clone GB11, BD, 560212), anti-CD107a (clone H4A, BioLegend, 328608), PINK1 antibody (Rockland, 600-401-GU5), anti-Rab4 antibody (EPR3043, Abcam, ab109009), goat anti-rabbit IgG secondary antibody, and APC (Invitrogen, A-10931).

Techniques: Expressing, Infection, Control, Activity Assay, Two Tailed Test