Journal: Neurotherapeutics

Article Title: Silmitasertib, an FDA-designated orphan CK2 inhibitor, ameliorates neuropathology and motor dysfunction in a Huntington's disease mouse model

doi: 10.1016/j.neurot.2026.e00859

Figure Lengend Snippet: CX-4945 decreases mHTT aggregation in symptomatic zQ175 mice. A) Schematic representation of experimental design for CX4945 treatment. B, C) Average bodyweight measurements for prodromal (B) and symptomatic (C) mice during 30-day oral gavage treatment. D) HTT (EM48) and GAPDH dot blots from striatal protein extracts of 6-month and 12-month CX-4945 and saline treated zQ175 mice. E, F) HTT (EM48) protein levels relative to GAPDH from images in D (n = 3 mice/group). G, H) HTT (EM48) immunostaining as a marker of mHtt aggregates and DAPI (stains nuclei) in dorsal striatum sections of 6-month (G) and 12-month (H) WT and zQ175 mice treated with CX4945 or saline. Scale bar, 50 μm. I, J) Quantification of HTT (EM48) + puncta per 0.4 mm 2 in the striatum of 6-month (I) and 12-month (J) WT and zQ175 mice treated with CX4945 or saline (n = 5–12 mice per group) analyzed from images in G and H respectively. Data was normalized to number of DAPI + cells and relativized to saline controls. K) HTT (EM48) and MAP2 immunostaining in dorsal striatum sections of 6-month and 12-month mice treated with CX4945 or saline neurons with DAPI (stains nuclei) in striatum of 6-month and 12-month CX4945 or saline. Scale bar, 5 μm. L, M) Quantification of HTT (EM48) + puncta colocalized with MAP2+ neurons in 6-month (L) and 12-month (M) zQ175 mice treated with CX4945 or saline (n = 10–12 mice/group for L, n = 6mice/group for M) from images in K. Data was normalized to number of DAPI + cells and relativized to saline controls. Error bars denote mean ± SEM. Student's unpaired t -test [E) t = .9292 dF = 4, F) t-3.204 dF = 4, L) t = 0.5951 dF = 20, M) t = 8.800 dF = 10] and ordinary one-way ANOVA with Tukey's multiple corrections [I) F = 29.99 dF = 3, J) F = 69.96 dF = 3]. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: The primary antibodies that were utilized and accompanying dilutions are: GFAP (chicken, ab5541, 1:2000), S100B (rabbit, Abcam ab41548, 1:500), EM48 (anti-HTT protein clone EM48) (mouse, mab5374, 1:500), GFP (chicken, Abcam AB13970, 1:1000), LAMP2 (rat, Thermo-Fisher, MA1-165, 1:100), Darpp32 (rat, R&D Systems MAB4230, 1:500), Iba1 (goat, Wako 011–27991, 1:500), cFos (Rabbit, Abcam ab190289, 1:500), MAP2 (Chicken, Abcam ab5392, 1:500) VGLUT1 (Guinea Pig, Millipore-Sigma AB5905, 1:500), VGLUT2 (Guinea Pig, Millipore-Sigma AB2251-I, 1:1000), PSD-95 (Rabbit, Thermofisher 51–6900, 1:500), LC3A (mouse, BIOSUSA bsm-33309 M, 1:1000), p62 (rabbit, Abcam ab91526, 1:500).

Techniques: Saline, Immunostaining, Marker

Journal: Neurotherapeutics

Article Title: Silmitasertib, an FDA-designated orphan CK2 inhibitor, ameliorates neuropathology and motor dysfunction in a Huntington's disease mouse model

doi: 10.1016/j.neurot.2026.e00859

Figure Lengend Snippet: CX-4945 treatment increases abundance of S100B + astrocytes and astrocytic HTT accumulation. A, B) S100B immunostaining in the dorsal striatum of 6-month (A) and 12-months (B) WT and zQ175 mice treated with CX4945 or saline treated. Scale bar, 50 μm. C, D) Quantification of S100B + cells in the dorsal whole striatum of 6-month (C) and 12-months (D) WT and zQ175 mice treated with CX4945 or saline (n = 4–12 mice/group) analyzed from images in A and B respectively. Data was normalized to number of DAPI + cells and relativized to saline WT. E) HTT (EM48)+ and S100B + immunostaining astrocytes within the dorsal striatum of 12-month zQ175 mice treated with CX4945 or saline. DAPI (stains nuclei) Scale bar, 5 μm. F, G) Quantification of HTT (EM48)/S100B colocalized puncta in 6-month (F) and 12-months (G) zQ175 mice treated with CX4945 or saline (n = 6–12mice/group). H, I) Quantification of cytoplasmic (H) and nuclear (I) HTT (EM48) + aggregates in S100B + astrocytes in 12-month zQ175 mice treated with CX4945 or saline (n = 6 mice/group) analyzed from images in E. Error bars denote mean ± SEM. Ordinary one-way ANOVA with Tukey's multiple corrections [C) F = 24.49 dF = 3, D) F = 18.43 dF = 3] and student's unpaired t -test [F) t = 0.5742 dF = 17.24, G) t = 2.341 dF = 10, H) t = 3.761 dF = 10, I) t-1.556 dF = 10]. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The primary antibodies that were utilized and accompanying dilutions are: GFAP (chicken, ab5541, 1:2000), S100B (rabbit, Abcam ab41548, 1:500), EM48 (anti-HTT protein clone EM48) (mouse, mab5374, 1:500), GFP (chicken, Abcam AB13970, 1:1000), LAMP2 (rat, Thermo-Fisher, MA1-165, 1:100), Darpp32 (rat, R&D Systems MAB4230, 1:500), Iba1 (goat, Wako 011–27991, 1:500), cFos (Rabbit, Abcam ab190289, 1:500), MAP2 (Chicken, Abcam ab5392, 1:500) VGLUT1 (Guinea Pig, Millipore-Sigma AB5905, 1:500), VGLUT2 (Guinea Pig, Millipore-Sigma AB2251-I, 1:1000), PSD-95 (Rabbit, Thermofisher 51–6900, 1:500), LC3A (mouse, BIOSUSA bsm-33309 M, 1:1000), p62 (rabbit, Abcam ab91526, 1:500).

Techniques: Immunostaining, Saline

Journal: Neurotherapeutics

Article Title: Silmitasertib, an FDA-designated orphan CK2 inhibitor, ameliorates neuropathology and motor dysfunction in a Huntington's disease mouse model

doi: 10.1016/j.neurot.2026.e00859

Figure Lengend Snippet: CX-4945 increases phagocytosis in S100B + astrocytes in symptomatic zQ175 mice. A) Left panels correspond to representative immunostaining of ex vivo slices from symptomatic zQ175 mice after incubation with FITC beads and CX4945 or saline. Immunostaining with S100B and DAPI (stains nuclei). Scale bar, 100 μm. Right panels (a and b) represent zoomed images from left panels. B) Percent of S100+ astrocytes with FITC beads in striatal slices from 12-month mice incubated with CX4945 or saline analyzed from images in A. 12 slices were obtained per group (n = 4 mice/group), slice averages are shown. Data was normalized to number of DAPI + cells and relativized to WT. C) Quantification of the number of FITC beads observed per S100B + astrocyte in striatal slices from 12-month mice incubated with CX4945 or saline analyzed from images in A. 12 slices were obtained per group (n = 4 mice/group), slice averages are shown. Data was normalized to number of DAPI + cells and relativized to WT. D) HTT (EM48), S100B and LAMP2 immunostaining in 12-month zQ175 mice treated with CX4945 or saline. E) Quantification of LAMP2+ puncta in S100B + astrocytes in 12-month zQ175 mice treated with CX4945 or saline (n = 6 mice/group). Analyzed from images in D. F) Percent of colocalized HTT (EM48)/LAMP2/S100B puncta analyzed from images in D (n = 6 mice/group). Error bars denote mean ± SEM. Student's unpaired t -test [B) t = 3.300 dF = 10.95, C) t = 5.490 dF = 13.96, E) t = 1.108 dF = 5.918, F) t = 3.121 dF = 10]. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001.

Article Snippet: The primary antibodies that were utilized and accompanying dilutions are: GFAP (chicken, ab5541, 1:2000), S100B (rabbit, Abcam ab41548, 1:500), EM48 (anti-HTT protein clone EM48) (mouse, mab5374, 1:500), GFP (chicken, Abcam AB13970, 1:1000), LAMP2 (rat, Thermo-Fisher, MA1-165, 1:100), Darpp32 (rat, R&D Systems MAB4230, 1:500), Iba1 (goat, Wako 011–27991, 1:500), cFos (Rabbit, Abcam ab190289, 1:500), MAP2 (Chicken, Abcam ab5392, 1:500) VGLUT1 (Guinea Pig, Millipore-Sigma AB5905, 1:500), VGLUT2 (Guinea Pig, Millipore-Sigma AB2251-I, 1:1000), PSD-95 (Rabbit, Thermofisher 51–6900, 1:500), LC3A (mouse, BIOSUSA bsm-33309 M, 1:1000), p62 (rabbit, Abcam ab91526, 1:500).

Techniques: Immunostaining, Ex Vivo, Incubation, Saline

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: Morelloflavone, a biflavonoid inhibitor of migration-related kinases, ameliorates atherosclerosis in mice.

doi: 10.1152/ajpheart.00669.2011

Figure Lengend Snippet: Fig. 5. Morelloflavone does not affect the status of plasma lipid peroxidation or phospholipase A2 (PLA2) enzymatic activities. A: assessment of plasma lipid peroxi- dation by thiobarbituric acid reactive substance (TBARS). NS, not statistically signif- icant. B–E: enzyme activity assays of secreted phospholipase A2 (types IB, IIA, and V) and lipoprotein-associated phospholipase (Lp-PLA2). IC50, the half maximal inhibitory concentration; MF, morelloflavone; T-PC, thioetheramide-1-palmitylthio-2- palmitoylamido-1,2-dideoxy-sn-glycero-3-phosphorylcholine (an inhibitor of sPLA2); MAFP, methyl arachidonyl fluorophosphonate (an inhibitor of Lp-PLA2; n 4). *P 0.05; #P 0.005.

Article Snippet: Recombinant human sPLA2 isoforms, group IB, group IIA, group V, and Lp-PLA2, were obtained from R&D Systems, and Cayman Chemical. sPLA2 enzyme activities in the presence of various concentrations of morelloflavone or other inhibitors were determined using a commercially available assay system according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Activity Assay, Concentration Assay

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Possible role for brain prostanoid pathways in the development of angiotensin II-salt hypertension in rats

doi: 10.1152/ajpregu.00535.2015

Figure Lengend Snippet: Phospholipase A2 and lipocalin prostaglandin D synthase gene expression using quantitative RT PCR in 4-day ANG II or vehicle-treated rats. A: PLA2 gene expression in PVN, NTS, SFO, RVLM, MCA, 3VCP, 4VCP, and OVLT. B: lipocalin prostaglandin D synthase gene expression in 4-day ANG II-treated rats in OVLT. #P < 0.05, significant overexpression after 4 days of ANG II infusion compared with vehicle treatment in rats. Between-group difference was analyzed by one-sample t-test with a hypothetical mean of 1.

Article Snippet: Blots prepared were then incubated overnight (4°C) with primary antibody anti-COX-1 (1:500; Millipore) or anti-tubulin (1:2,000 dilution; Millipore) or PGDs (Cayman Chemicals) or phospholipase A2 (PLA2) (Novus Biologicals).

Techniques: Gene Expression, Quantitative RT-PCR, Over Expression

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Possible role for brain prostanoid pathways in the development of angiotensin II-salt hypertension in rats

doi: 10.1152/ajpregu.00535.2015

Figure Lengend Snippet: Cytosolic phospholipase A2, cyclooxygenase-1, and lipocalin prostaglandin D synthase protein expression in 4-day ANG II or vehicle-treated rats. A: cytosolic PLA2 protein expression in 4-day ANG II-treated rats in middle cerebral artery (vehicle; n = 7, ANG II; n = 5). B: COX-1 protein expression in 4-day ANG II-treated rats in middle cerebral artery (vehicle; n = 3, ANG II; n = 5). C: lipocalin-type prostaglandin D synthase protein expression in 4-day ANG II-treated rats in CP in third ventricle (vehicle; n = 6, ANG II; n = 4). #Overexpression of proteins (P < 0.05) in ANG II infusion day 4 compared with control rats.

Article Snippet: Blots prepared were then incubated overnight (4°C) with primary antibody anti-COX-1 (1:500; Millipore) or anti-tubulin (1:2,000 dilution; Millipore) or PGDs (Cayman Chemicals) or phospholipase A2 (PLA2) (Novus Biologicals).

Techniques: Expressing, Over Expression, Control

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Possible role for brain prostanoid pathways in the development of angiotensin II-salt hypertension in rats

doi: 10.1152/ajpregu.00535.2015

Figure Lengend Snippet: Cytosolic phospholipase A2, cyclooxygenase-1, prostaglandin D synthase protein expression in 4-day ANG II compared to vehicle-treated rats

Article Snippet: Blots prepared were then incubated overnight (4°C) with primary antibody anti-COX-1 (1:500; Millipore) or anti-tubulin (1:2,000 dilution; Millipore) or PGDs (Cayman Chemicals) or phospholipase A2 (PLA2) (Novus Biologicals).

Techniques: Expressing, Control

Journal: Biology of reproduction

Article Title: PGE2 increases inflammatory damage in Escherichia coli-infected bovine endometrial tissue in vitro via the EP4-PKA signaling pathway.

doi: 10.1093/biolre/ioy162

Figure Lengend Snippet: Figure 1. E. coli infection of bovine endometrial explants induced PLA2, AA, COX-2 and mPGES-1 expression, resulting in PGE2 and CysLT accumulation. mRNA and protein expression of (A, B) PLA2, (C) COX-2, and (D) mPGES-1 in bovine endometrial explants treated with MK886, MF63, CAY10404, or NS398 (10-6 –10-5

Article Snippet: Concentrations of PLA2, AA, PGE2, leukotrienes, IL1β, IL6, IL8, and TNF-α were measured in tissue culture supernatants and tissue extracts with ELISAs (cPLA2 Assay kit 765021, Cayman; Bovine Arachidonic Acid ELISA Kit CSB-EQ027590BO, Cusabio; Prostaglandin E 2 ELISA Kit-Monoclonal 514010, Cayman; Cysteinyl Leukotriene (CysLT) ELISA Kit 500390, Cayman; Bovine IL-1 beta ELISA Reagent Kit ESS0027, Kingfisher Biotech; Bovine IL-6 ELISA Reagent Kit DY8190, R&D Systems; Bovine Interleukin 8 (IL-8) ELISA Kit CSB-E130528, Cusabio; and Bovine TNF-alpha Duo Set DY2279, R&D Systems Europe Ltd), following the manufacturers' instructions.

Techniques: Infection, Expressing

Journal: Molecular Neurodegeneration

Article Title: sPLA2-IIA modifies progranulin deficiency phenotypes in mouse models

doi: 10.1186/s13024-025-00863-8

Figure Lengend Snippet: PGRN physically interacts with sPLA2-IIA and regulates sPLA2-IIA levels and activity. ( a ) Schematic illustration of the SILAC experiment searching for PGRN interactors in HEPG2 cells. Proteins with more than 4 peptides and p-value < 0.01 and Log2 (GFP/GFP-PGRN) ratio <-1 were labeled in green. ( b ) Human PGRN and sPLA2-IIA interact when overexpressed in HEK293T cells. Human sPLA2-IIA-myc and human PGRN-expressing constructs were transfected into HEK293T cells as indicated. Lysates and conditioned medium were prepared 3 days later and immunoprecipitated with anti-Myc antibodies. ( c ) Mouse-sPLA2-IIA-myc and FLAG-mouse-PGRN expressing constructs were transfected into HEK293T cells as indicated. Lysates and conditioned medium were prepared 3 days later and immunoprecipitated with anti-Myc antibodies. ( d ) PGRN inhibits sPLA2-IIA activities in vitro. Recombinant sPLA2-IIA protein was incubated with substrates in the absence or presence of recombinant PGRN at a 1:1 or 1:2 molar ratio, and sPLA2-IIA activities were measured. Data are presented as mean ± SEM from three independent experiments ( n = 3). *, p < 0.05; **, p < 0.01, Student’s t-test. ( e ) The levels of sPLA2-IIA in tissue lysates from 2-month-old FVB mice were analyzed by western blot. ( f ) The levels of sPLA2-IIA are upregulated in the duodenum in PGRN-deficient mice. The duodenum of 2-month-old WT B6 mice and WT ( Pla2g2a +/+ Grn +/+ ) and Grn −/− ( Pla2g2a +/+ Grn −/− ) mice in the FVB background were lysed, and the same amount of the total protein was used for western blot to measure sPLA2-IIA levels. sPLA2-IIA levels in the mouse duodenum were quantified. Data are presented as mean ± SEM from 5 mice per group ( n = 5). *, p < 0.05, Student’s t-test. ( g , h ) Serum levels of prostaglandin and C18:1, C18:2, and C18:3 fatty acids are upregulated in PGRN-deficient mice. Prostaglandin (predominantly PGE2 and PGD2, which have the same mass) concentration in serum and cortical lysates was measured by mass spectrometry using PGE2 as the standard. The levels of C18:1, C18:2, and C18:3 fatty acids were measured by mass spectrometry, and the relative abundance in WT and Grn −/− mice was shown. Data are presented as mean ± SEM from six mice per group ( n = 6). *, p < 0.05, **, p < 0.01, ***, p < 0.001, and ****, p < 0.0001, Student’s t-test

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Activity Assay, Multiplex sample analysis, Labeling, Expressing, Construct, Transfection, Immunoprecipitation, In Vitro, Recombinant, Incubation, Western Blot, Concentration Assay, Mass Spectrometry

Journal: Molecular Neurodegeneration

Article Title: sPLA2-IIA modifies progranulin deficiency phenotypes in mouse models

doi: 10.1186/s13024-025-00863-8

Figure Lengend Snippet: sPLA2 inhibitor treatment rescues PGRN deficiency phenotypes in mice. 5-week-old Pla2g2a +/+ Grn +/+ and Pla2g2a +/+ Grn −/− mice in FVB/B6 mixed background were treated with corn oil control or sPLA2 inhibitors, LY333013 or KH064 for 3 weeks. ( a ) Measurement of total sPLA2 activity in the serum and cortical lysates from untreated and LY333013 or KH064-treated mice. Data are presented as mean ± SEM from five mice per group ( n = 5). *, p < 0.05; **, p < 0.01, Student’s t-test. ( b ) Brain sections were immunostained with anti-GFAP, CD68, and IBA1 antibodies, and representative images from the cortex region were shown (Scale bar = 100 μm). Another set of brain sections was stained with Hoechst and auto-fluorescent lipofuscin signals (red) in the thalamus region were imaged (scale bar = 10 μm). ( c ) Quantification of GFAP, CD68, and IBA1 levels and auto-fluorescent lipofuscin signals for the experiment in (b). Data are presented as mean ± SEM from five mice per group ( n = 5). 3 sections were analyzed for each mouse brain. *, p < 0.05; **, p < 0.01, Student’s t-test. ( d ) Brain sections were immunostained with anti-CathD, NeuN, and IBA1 antibodies, and representative images from the cortex region were shown (Scale bar = 10 μm). ( e ) CathD signals in IBA1-positive microglia and NeuN-positive neurons were quantified. 10–20 confocal images were randomly captured from each brain section. Ten to twenty microglia and fifty to sixty neurons were quantified per section. 3 sections were analyzed for each mouse brain. Data are presented as mean ± SEM from 3–4 mice per group ( n = 3–4). *, p < 0.05; **, p < 0.01, Student’s t-test

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Control, Activity Assay, Staining

Journal: Molecular Neurodegeneration

Article Title: sPLA2-IIA modifies progranulin deficiency phenotypes in mouse models

doi: 10.1186/s13024-025-00863-8

Figure Lengend Snippet: AAV9-mediated overexpression of sPLA2-IIA leads to enhanced glial activation in 2-month-old B6 Grn −/− mice. ( a , b ) Expression of sPLA2-IIA at endogenous levels in Grn −/− B6 mice does not affect glial activation and lipofuscin accumulation. Immunostaining of GFAP, CD68, and IBA1 and autofluorescent lipofuscin signals in brain sections from 2-month-old Pla2g2a −/− Grn −/− and Pla2g2a +/+ Grn −/− littermate mice in the B6 background. Representative images from the cortex and the thalamus were shown for GFAP/CD68/IBA1 staining and lipofuscin signals. Scale bar = 100 μm (for GFAP/CD68/IBA1) and 10 μm (for lipofuscin). GFAP, CD68, and IBA1 levels and auto-fluorescent lipofuscin signals were quantified in ( b ). Data are presented as mean ± SEM from 4 mice per group ( n = 4). 3 brain sections were analyzed for each mouse brain. ( c , d ) AAV9-mediated overexpression of sPLA2-IIA leads to enhanced glial activation in 2-month-old Grn −/− mice in B6 mice. B6 WT ( Pla2g2 −/− Grn +/+ ) and Grn −/− ( Pla2g2a −/− Grn −/− ) pups at postnatal day (P0) were injected with AAV9-sPLA2-IIA or the buffer control, and the mice were collected at 2 months old. ( c ) Brain sections were immunostained with anti-GFAP, CD68, and IBA1 antibodies, and representative images from the cortex and thalamus regions were shown (Scale bar = 100 μm). ( d ) Quantification of GFAP, CD68, and IBA1 levels for the experiment in (c). Data are presented as mean ± SEM from three to five mice per group ( n = 3–5). Three sections were analyzed for each mouse brain. *, p < 0.05; **, p < 0.01; ****, p < 0.0001, Student’s t-test

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Over Expression, Activation Assay, Expressing, Immunostaining, Staining, Injection, Control

Journal: Molecular Neurodegeneration

Article Title: sPLA2-IIA modifies progranulin deficiency phenotypes in mouse models

doi: 10.1186/s13024-025-00863-8

Figure Lengend Snippet: AAV9-mediated overexpression of sPLA2-IIA leads to lipofuscin accumulation and lysosomal enlargement in microglia in 2-month-old B6 Grn −/− mice. B6 WT ( Pla2g2 −/− Grn +/+ ) and Grn −/− ( Pla2g2a −/− Grn −/− ) pups at postnatal day (P0) were injected with AAV9-sPLA2-IIA or the buffer control, and the mice were collected at 2 months old. ( a ) Brain sections were stained with NeuN and IBA1, and auto-fluorescent lipofuscin signals (blue) in the cortex region were imaged (scale bar = 10 μm). ( b ) Quantification of autofluorescent lipofuscin signals in the cortex. Data are presented as mean ± SEM from three to five mice per group ( n = 3–5). Three sections were analyzed for each mouse brain. *, p < 0.05; **, p < 0.01; ****, p < 0.0001, Student’s t-test. ( c ) Brain sections were immunostained with anti-CathD, NeuN, and IBA1 antibodies, and representative images from the cortex region were shown (Scale bar = 10 μm). ( d ) CathD signals in IBA1-positive microglia and NeuN-positive neurons were quantified. 10–20 confocal images were randomly captured from each brain section. Fifteen to thirty microglia and fifty to sixty neurons were quantified per Sect. 3 sections were analyzed for each mouse brain. Data are presented as mean ± SEM from 3–5 mice per group ( n = 3–5). *, p < 0.05; **, p < 0.01; ****, p < 0.0001, Student’s t-test

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Over Expression, Injection, Control, Staining

Journal: bioRxiv

Article Title: Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation

doi: 10.1101/2023.04.06.535844

Figure Lengend Snippet: ( A ) Schematic illustration of the SILAC experiment searching for PGRN interactors in HEPG2 cells. Proteins with more than 4 peptides and p value <0.01 and Log2 (GFP/GFP-PGRN) ratio <−1 were labeled in green. ( B ) Human PGRN and sPLA2-IIA interact when overexpressed in HEK293T cells. Human sPLA2-IIA-myc and human PGRN expressing constructs were transfected into HEK293T cells as indicated. Lysates and conditioned medium were prepared 3 days later and immunoprecipitated with anti-Myc antibodies. ( C ) Mouse-sPLA2-IIA-myc and FLAG-mouse-PGRN expressing constructs were transfected into HEK293T cells as indicated. Lysates and conditioned medium were prepared 3 days later and immunoprecipitated with anti-Myc antibodies. ( D ) Human PGRN interacts with human sPLA2-IIA through granulin F and G. Human-sPLA2-IIA-myc and GFP-PGRN / granulin constructs were transfected into HEK293T cells as indicated. Lysates were prepared 2 days later and immunoprecipitated with GFP-Trap. ( E ) Diagram of PGRN structure. ( F) Deletion of granulin F and G reduces the interaction between PGRN and sPLA2-IIA. Untagged human sPLA2-IIA and Flag-PGRN / Flag-PGRNΔGF constructs were transfected into HEK293T cells as indicated. Lysates and conditioned medium were collected 2 days later and immunoprecipitated with anti-FLAG antibodies.

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Labeling, Expressing, Construct, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation

doi: 10.1101/2023.04.06.535844

Figure Lengend Snippet: ( A ) PGRN inhibits sPLA2-IIA activities in vitro . Recombinant sPLA2-IIA protein was incubated with substrates in the absence or presence of recombinant PGRN at a 1:1 or 1:2 molar ratio and sPLA2-IIA activities were measured. ( B, C ) Control and PGRN deficient Raw264.7 cells were cultured in a serum-free medium for 24 hours before the lysates and conditioned media (CM) were harvested. Recombinant PGRN protein was added to the medium at 5ug/ml when indicated. Proteins from the CM were precipitated with TCA. Cell lysates and TCA precipitated medium were blotted with anti-sPLA2-IIA-antibodies. The levels of sPLA2-IIA in the cell lysate and medium were quantified and normalized to GAPDH and transferrin, respectively. The value in PGRN deficient conditions was then normalized to that in the control cells. ( D ) Pla2g2a mRNA levels are not affected by PGRN loss. mRNAs were extracted from control and PGRN-deficient Raw264.7 cells. The mRNA levels of sPLA2-IIA were quantified by RT-qPCR and normalized to β-actin. Data are presented as mean ± SEM from three independent experiments. *, p<0.05; **, p<0.01, Student’s t-test (for two groups comparison) or one-way ANOVA tests (for multiple comparisons).

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: In Vitro, Recombinant, Incubation, Control, Cell Culture, Quantitative RT-PCR, Comparison

Journal: bioRxiv

Article Title: Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation

doi: 10.1101/2023.04.06.535844

Figure Lengend Snippet: ( A ) Immunostaining of GFAP, CD68, and IBA1 and auto-fluorescent lipofuscin signals in brain sections from 2-month-old Pla2g2a +/+ and Pla2g2a +/+ Grn −/− mice. Representative images from the cortex were shown for GFAP/CD68/IBA1 staining and images from the thalamus were shown for lipofuscin signals. Scale bar =100 µm (GFAP/CD68/IBA1); Scale bar =10 µm (Lipofuscin images). ( B ) Quantification of GFAP, CD68, and IBA1 levels and auto-fluorescent lipofuscin signals for the experiment in (a). Data are presented as mean ± SEM from 5 mice of mixed sexes per group (n=5). 3 brain sections were analyzed for each mouse brain. **, p<0.01, Student’s t-test.

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Immunostaining, Staining

Journal: bioRxiv

Article Title: Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation

doi: 10.1101/2023.04.06.535844

Figure Lengend Snippet: ( A, B ) Total RNAs were extracted from the cortex of 5-month-old Pla2g2a +/+ and Pla2g2a +/+ Grn −/− mice and the RNA-seq was performed to analyze gene expression changes (n=3). Differential expressed genes (DEGs) with FDR P ≤ 0.05, absolute Log2FC ≥ 0.5 between Pla2g2a +/+ and Pla2g2a +/+ Grn −/− groups are plotted using Heatmapper (A; Upregulated in Grn −/− ; B: downregulated in Grn −/− ). Inflammation genes and lysosomal genes are highlighted in red and green, respectively. ( C ) GSEA pathway analysis of the upregulated pathways with NES (normalized enrichment score)>1.0, NOM p-val (nominal p-value)<0.05, and FDR q-val (false discovery rate q value)<0.25 in RNA samples from the cortex of Pla2g2a +/+ Grn −/− mice compared to these from Pla2g2a +/+ mice.

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation

doi: 10.1101/2023.04.06.535844

Figure Lengend Snippet: ( A, B ) Immunostaining of Cathepsin D (CathD), LAMP1, and IBA1 in brain sections from 2-month-old Pla2g2a +/+ and Pla2g2a +/+ Grn −/− mice. Representative images from the cortex were shown. CathD and LAMP1 signals in IBA1-positive microglia were quantified. 3 sections per mouse brain and 3 mouse brains were used for each genotype. 10–12 cells per section were analyzed. Scale bar =10 µm. n=3, **, p<0.01. ( C ) Immunostaining of CathD, GFAP, and NeuN in brain sections from 2-month-old Pla2g2a +/+ and Pla2g2a +/+ Grn −/− mice. Representative images from the cortex were shown. Similar results were obtained from 3 mouse brains. Scale bar=8µm

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation

doi: 10.1101/2023.04.06.535844

Figure Lengend Snippet: 5-week-old Pla2g2a +/+ and Pla2g2a +/+ Grn −/− mice were treated with corn oil control or PLA2-IIA inhibitors, LY333013 or KH064 for 3 weeks. ( A ) Brain sections were immunostained with anti-GFAP, CD68, and IBA1 antibodies, and representative images from the cortex region were shown (Scale bar=100 µm). Another set of brain sections was stained with Hoechst and auto-fluorescent lipofuscin signals (red) in the thalamus region were imaged (scale bar=10µm). ( B ) Quantification of GFAP, CD68, and IBA1 levels and auto-fluorescent lipofuscin signals for the experiment in (A). Data are presented as mean ± SEM from five mice per group (n=5). 3 sections were analyzed for each mouse brain. *, p<0.05; **, p<0.01, one-way ANOVA.

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Control, Staining

Journal: bioRxiv

Article Title: Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation

doi: 10.1101/2023.04.06.535844

Figure Lengend Snippet: 5-week-old Pla2g2a +/+ and Pla2g2a +/+ Grn −/− mice were treated with corn oil control or PLA2-IIA inhibitors, LY333013 or KH064 for 3 weeks. ( A ) Brain sections were immunostained with anti-CathD, NeuN, and IBA1 antibodies, and representative images from the cortex region were shown (Scale bar=10 µm). ( B ) CathD signals in IBA1 positive microglia and NeuN positive neurons were quantified. 10–20 confocal images were randomly captured from each brain section. Ten to twenty microglia and fifty to sixty neurons were quantified per section. 3 sections were analyzed for each mouse brain. Data are presented as mean ± SEM from 3–4 mice per group (n=3–4). *, p<0.05; **, p<0.01, one-way ANOVA.

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Control

Journal: bioRxiv

Article Title: Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation

doi: 10.1101/2023.04.06.535844

Figure Lengend Snippet: ( a ) sPLA2-IIA levels were measured in the lysates from postmortem human brain tissues from healthy controls and FTLD with GRN mutations or Tau aggregation using ELISA. Data are presented as mean ± SEM from 4–6 cases per group (n=4–6). *, p<0.05, Student’s t-test. ( b ) Anterior orbital gyrus tissues from healthy controls and FTLD- GRN patients were immunostained with anti-sPLA2-IIA and GFAP antibodies. Representative confocal images from 3 different cases for each genotype were shown (Scale bar=10 µm).

Article Snippet: Mouse Pla2g2a -Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc).

Techniques: Enzyme-linked Immunosorbent Assay