Journal: Journal of Functional Foods

Article Title: Krill oil attenuates diabetes-associated cognitive dysfunction by inhibiting microglial polarization-induced neuron injury

doi: 10.1016/j.jff.2024.106064

Figure Lengend Snippet: Fig. 5. KO had no protective effect on AGEs-activated neuron apoptosis. (A) Diagram for culture and treatment of N2a cells; (B) TUNEL staining with (C) percentage of positive cells quantified; (D) protein levels of BAX, BCL2 and cleaved Caspase3. The data were summarized as means ± SD (n = 3). *P < 0.05 vs. Ctrl; #P < 0.05 vs. AGEs + Veh. Abbreviations: AGEs, advanced glycation end products; Veh, vehicle (glycerol). Other abbreviations are the same as in Fig. 4.

Article Snippet: Mouse neuron N2a cells were obtained from The American Type Culture Collection (ATCC, CCL131, Manassas, Virginia, USA).

Techniques: TUNEL Assay, Staining

Journal: Nature Communications

Article Title: Localized molecular chaperone synthesis maintains neuronal dendrite proteostasis

doi: 10.1038/s41467-024-55055-7

Figure Lengend Snippet: a Schematic of the pulldown strategy used to identify RBPs binding to the Hspa8 3′ UTR, and a table of the RBPs identified as specifically bound to the Hspa8 3′ UTR in extracts from Ctrl (black *) and MG132-stressed (red *) N2A cells by MS (Created in BioRender. ALAGAR, L. (2024) https://BioRender.com/f90a722 ). Proteomics data is provided in Source data. b Pulldown experiments to validate the binding of STAU2 and FUS to the Hspa8 3′ UTR were analyzed by western blot in two independent replicates. I input, PD pulldown. c , d Representative images of primary mouse motor neurons expressing GFP (Ctrl) or GFP and shRNAs against STAU2 ( c ) or FUS ( d ). Three days after microinjection, stress was induced with 10 μM MG132 for 7 h, and Hspa8 mRNA expression was detected by smFISH. Scale bars = 5 μm. e – h Quantification of the densities of H s pa8 ( e , g ) and eEf1a1 ( f , h ) mRNAs per pixel of soma or dendrite area in Ctrl and MG132-stressed motor neurons expressing GFP with and without the indicated shRNA expression plasmids. For STAU2, the data are the mean ± SEM of three independent experiments (neurons GFP control n = 33, GFP MG132 n = 27, STAU2 shRNA control n = 18, STAU2 shRNA MG132 n = 23; dendrites GFP control n = 105, GFP MG132 n = 111, STAU2 shRNA control n = 72, STAU2 shRNA MG132 n = 98; dots indicate individual soma and dendrite values). P values for Hspa8 mRNA (**** P < 0.0001, ns no significant ( P = 0.99)) and for eEf1a1 mRNA (Soma: ** P < 0.01 (0.031), ns no significant ( P = 0.26), Dendrites: * P < 0.05 (0.076), ns no significant ( P = 0.98). For FUS, five independent experiments (neurons GFP control n = 69, GFP MG132 n = 72, FUS shRNA control n = 75, FUS shRNA MG132 n = 69; dendrites GFP control n = 246, GFP MG132 n = 232, FUS shRNA control n = 255, FUS shRNA MG132 n = 224). P values for Hspa8 mRNA (Soma: ns no significant ( P = 0.88 in Ctrl and 0.1 in MG132), Dendrites: *** P < 0.001; ns no significant ( P = 0.905)) and for eEf1a1 mRNA (Soma: ns no significant ( P = 0.99 in Ctrl and MG132), Dendrites: ns no significant ( P = 0.99 in Ctrl and 0.34 in MG132)) (by 1-way ANOVA). i Ratio of the dendrite to soma Hspa8 mRNA density calculated by averaging the number of mRNAs/pixel of all dendrites of a neuron and dividing it by the number of mRNA/pixel of soma. The data are the mean ± SEM of three independent experiments (GFP control n = 70, GFP MG132 n = 72, FUS shRNA control n = 75, FUS shRNA MG132 n = 69 neurons) from G. * P < 0.05 ( P = 0.021); ns no significant ( P = 0.13) (by 1 way ANOVA). j Representative dendrites from Ctrl and MG132-stressed motor neurons expressing the proteostasis reporter plasmid FLUC-GFP with and without FUS knockdown. GFP aggregation is proportional to proteostasis loss. Scale bar = 10 μm. k Quantification of the GFP signal granularity (the coefficient of variation) in each dendrite in I. The data are the mean ± SEM of four independent experiments (GFP control n = 97, GFP MG132 n = 138, FUS shRNA control n = 88, FUS shRNA MG132 n = 115 dendrites). **** P < 0.0001;** P < 0.01 ( P = 0.0021) (by 1 way ANOVA). Source data are provided as a Source Data file.

Article Snippet: HSPA8 3 ′ UTR PP7-HSPA8 and PP7-LacZ RNA were first PCR amplified from N2A genomic DNA or a plasmid (donated by Dr. Jerry Pelletier) using the primers listed below, and then in vitro transcribed with a MEGAshortscriptTM T7 Transcription Kit (Invitrogen, #AM1354) following the manufacturer’s instructions.

Techniques: Binding Assay, Western Blot, Expressing, Microinjection, shRNA, Control, Plasmid Preparation, Knockdown

Journal: bioRxiv

Article Title: Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

doi: 10.1101/2020.12.13.417964

Figure Lengend Snippet: (A and C ) PJA1 transcript level was decreased upon over-expression of ataxin-3 and huntingtin protein. Normal and polyglutamine (polyQ) expanded ataxin-3 (A) and huntingtin (C) plasmids and their corresponding empty vectors pEGFP-C1 and pDsRED-C1 were transfected in mouse neuroblastoma, Neuro2A cells. 72 hours after transfection, total RNA was isolated from the cells and subjected to quantitative real-time PCR. Data were collected from three separate experiments and normalised to the levels of GAPDH. Values are the mean ± S.D. Significance of the data was calculated by t-test. p <0.05 for *, p<0.01 for **, p<0.001 for *** compared to non-transfected cells. (B and D ) PJA1 protein level is reduced in ataxin-3 and huntingtin transformed cells. The lysates from (A) and (C) over-expressing ataxin-3 and huntingtin respectively were immunoblotted with PJA1 and GAPDH antibody. Lysate shows the expression of ataxin-3 and huntingtin protein by GFP and DsRed antibody. The bands were quantified, normalized with GAPDH and plotted as bar diagram. Error bar represents SD of three independent experiments. Significance was calculated by t-test, *(p<0.05), **(p<0.01), *** (p<0.001). NT stands for non-transfected cells.

Article Snippet: Human Embryonic Kidney 293T (HEK293T), HEK293 or Mouse Neuroblastoma (N2A) cells were obtained from National Centre for Cell Science (NCCS), Pune, India.

Techniques: Over Expression, Transfection, Isolation, Real-time Polymerase Chain Reaction, Transformation Assay, Expressing

Journal: bioRxiv

Article Title: Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

doi: 10.1101/2020.12.13.417964

Figure Lengend Snippet: ( A-E) PJA1 interacts with ataxin-3 proteins. (A) PJA1 does not interact with GFP protein. GFP empty vector (EV) and HA-PJA1 constructs were co-transfected in HEK293T cells. Cell lysate was immunoprecipitated with either anti-GFP or anti-HA antibody followed by western blotting and probing with anti-HA and anti-GFP antibody respectively. (B-D) 20Q truncated (20QT), 80Q truncated (80QT) and 130Q full length (130QF) ataxin-3 in pEGFP-N1 vector were co-transfected with HA-tagged PJA1 (pCMV-HA-PJA1) in HEK293T cells (A and B) and Neuro2A cells (C and D) respectively. 24 hours after transfection, cells were lysed and subjected to immunoprecipitation either with anti-HA antibody (A and C) or with anti-GFP antibody (B and D). The blots were consecutively probed with anti-GFP and anti-HA antibody for detection of ataxin-3 and PJA1 respectively. The total lysate shows the expression of the respective proteins. (F and G) PJA1 interacts with endogenous ataxin-3 protein. HA-tagged PJA1 and ΔRING-PJA1 were transfected in HEK293T cells. 24 hours after transfection, cells were lysed and subjected to immunoprecipitation using anti-SCA3 (F) or anti-HA antibody (G). The blots were consecutively probed with respective antibodies. The total lysate shows the expression of the respective proteins. (H and I) PJA1 interacts with huntingtin protein. N-terminal fragment of 16Q huntingtin (16QHTT) protein in pEGFP-C1 vector (H) and 83QHTT in pDsRed vector (I) were co-transfected with HA-PJA1 in HEK293T cells. Cells were harvested and processed for immunoprecipitation with anti-HA antibody. The blots were successively probed with anti-GFP/anti-DsRed and anti-HA antibody. * indicates non-specific protein. GAPDH is the loading control for all the experiments. Results are representative of three independent experiments. NT and EV represent non-transfected cells and empty vector respectively.

Article Snippet: Human Embryonic Kidney 293T (HEK293T), HEK293 or Mouse Neuroblastoma (N2A) cells were obtained from National Centre for Cell Science (NCCS), Pune, India.

Techniques: Plasmid Preparation, Construct, Transfection, Immunoprecipitation, Western Blot, Expressing

Journal: bioRxiv

Article Title: Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

doi: 10.1101/2020.12.13.417964

Figure Lengend Snippet: (A) shRNA mediated down regulation of PJA1 transcript level. Neuro2A cells were transiently transfected with short hairpin RNA (shRNA) targeted to PJA1. Cells were harvested after 48 hours. Total RNA was isolated and subjected to quantitative real-time PCR. Data were collected from three independent experiments and normalised to the levels of GAPDH. Values are the mean ± SD. Significance was calculated by t-test, p< 0.01. (B) PJA1 down regulation enhances the level of ataxin-3 proteins in Neuro2A cells. The protein level of 20QT, 80QT and 130QF ataxin-3 was assayed in PJA1 silenced condition by western blot analysis. The blots were probed with anti-GFP antibody for detection of ataxin-3. GAPDH is the loading control. The bands were quantified, normalized with GAPDH and plotted as bar diagram. Error bar represents SD of three independent experiments. Significance was calculated by t-test, *, ** indicate p<0.05 and p<0.01 respectively. (C and D) Downregulation of PJA1 enhances ataxin-3 and HTT aggregates. 80QT ataxin-3 (C) or 83QHTT (D) plasmids were transfected in Neuro2A cells upon silencing of PJA1 with shRNA. After 48 hours cells were viewed under fluorescence microscopy. The aggregates were counted and plotted in a bar graph. Values are the mean ± SD, Significance was calculated by t-test, p<0.01. Same cells were processed for immunoblotting and the blots were probed with anti-GFP, anti-DsRed and anti-GAPDH antibodies. The 83QHTT bands were similarly quantified and plotted as bar diagram, the error bar representing SD of three independent experiments. Significance was calculated by t-test, p<0.05. (E) siRNA mediated down regulation of PJA1 transcript level. Neuro2A cells were transiently transfected with scramble or siRNA targeted to PJA1. Cells were harvested after 48 hours. Total RNA was isolated and subjected to quantitative real-time PCR. Data were collected from three independent experiments and normalised to the levels of GAPDH. Values are the mean ± SD. Significance was calculated by t-test, p< 0.01. (F) siRNA mediated down-regulation of PJA1enhances ataxin-3 protein level in Neuro2A cells. The protein level of 20QT (Fi), 80QT (Fii) ataxin-3 was assayed in PJA1 silenced condition by western blot analysis. The blots were probed with anti-GFP antibody for detection of ataxin-3. GAPDH is the loading control. The bands were quantified and normalized with GAPDH and plotted as a bar diagram. Error bars represent a cumulative of three experiments performed independently. Significance was calculated by t-test, p<0.05. (G) The same cells were viewed under fluorescence microscope, where enhanced overall fluorescence and aggregation in PJA1 silenced condition was observed for 20QT and 80QT ataxin-3 respectively.

Article Snippet: Human Embryonic Kidney 293T (HEK293T), HEK293 or Mouse Neuroblastoma (N2A) cells were obtained from National Centre for Cell Science (NCCS), Pune, India.

Techniques: shRNA, Transfection, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

doi: 10.1101/2020.12.13.417964

Figure Lengend Snippet: (A-C) Time and dose dependent expression of PJA1 enhances degradation of ataxin-3 protein. Neuro2A cells were transiently transfected with 80QT (A), and 130QF (B) with varying concentrations of PJA1 as indicated. 24 hours after transfection, ataxin-3 level was detected in cell lysates by western blotting with anti-GFP antibody. C. 80QT ataxin-3 and PJA1 (3 μg) were co-transfected in Neuro2A cells and the cells were collected at varying time as indicated. Western blot analysis shows ataxin-3 level. GAPDH and Tubulin was used as loading controls. (D and E) PJA1 facilitates degradation of 80QT ataxin-3 in mammalian and yeast system. (D) GAL-shut off chase was performed to measure the turn-over of ataxin-3. PJA1 expression was carried out with addition of copper followed by induction of ataxin-3 with 2% galactose for 2 hours. The expression of ataxin-3 was shut off using 2% glucose, and chased for indicated times. Ataxin-3 level was detected by western blotting with anti-GFP antibody. PGK1 is loading control. Data were collected from three separate experiments and normalised to the levels of PGK1. The band intensities were quantified using imageJ software and plotted in the adjacent graph. Error bar indicates SD. (E) Cycloheximide chase of 80QT ataxin-3 was done upon silencing of PJA1 with smartpool siRNA (Dharmacon) targeted to PJA1 in Neuro2A cells as described in method section. 10μg/ml of cycloheximide was used to stop translation and chased for 10 hours. Ataxin-3 level was detected by western blotting with anti-GFP antibody. GAPDH acts as the loading control. (F-H) PJA1 promotes ubiquitination of ataxin-3. (F and G) 80QT and 20QT ataxin-3 plasmids were transformed along with PJA1 and myc-ubiquitin into WT (F) and atg8Δ (G) yeast cell respectively. Expression of PJA1 was induced with 300μM copper for 2 hours prior to ataxin-3 induction with 2% galactose for 4 hours. Yeast cells were then harvested and immunoprecipitation was performed using anti-GFP antibody. The blots were probed with anti-myc and anti-GFP antibody for detection of ubiquitinated ataxin-3 and the amount of immunoprecipitated ataxin-3 respectively. Lysate shows the expression of the proteins. PGK1 was the loading control. (H) Silencing of PJA1 reduces ubiquitination of 80QT ataxin-3. PJA1 was silenced in Neuro2A cells either with shRNA or siRNA targeted to PJA1. GFP-80QT ataxin-3 and HA-ubiquitin plasmids were co-transfected and the ubiquitination of 80QT ataxin-3 was detected upon immunoprecipitating ataxin-3 with anti-GFP antibody followed by western blotting with anti-HA antibody. Lysates show the expression of respective proteins. GAPDH is the loading control.

Article Snippet: Human Embryonic Kidney 293T (HEK293T), HEK293 or Mouse Neuroblastoma (N2A) cells were obtained from National Centre for Cell Science (NCCS), Pune, India.

Techniques: Expressing, Transfection, Western Blot, Software, Transformation Assay, Immunoprecipitation, shRNA