Journal: EMBO Reports

Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol

doi: 10.1038/s44319-024-00328-x

Figure Lengend Snippet: ( A ) Immunoblot analysis of SseI-interacting proteins (IQGAP, TRIP6, and ARF1) on isolated peroxisomes from STM-infected (MOI = 10) and uninfected HeLa cells, with whole cell lysate (WCL). The representative blot was probed with anti-IQGAP, anti-TRIP6, and anti-ARF1 antibodies. ( B ) Immunoblot analysis of proteins after SseI pulldown from HeLa cells expressing HA-tagged SseI WT and SseI ΔGKM. Cell lysates (input) were used for SseI pulldown with anti-HA antibody. IgG was used as a control. The representative blot was probed with anti-HA, anti-ARF1, and anti-PEX5 antibodies. ( C ). Immunoblot analysis of in vitro binding between purified His-tagged SseI and HA-tagged ARF1 in HeLa whole cell lysates (WCL). The representative blot was probed with anti-His and anti-HA antibodies. ( D ) Immunoblot analysis of Active ARF1 was pulled down from WT, Δ sseI STM-infected (MOI = 10), and uninfected HeLa cells. Immunoblot analysis was performed to detect active ARF1. Ponceau staining was used as a loading control for the pulldown samples. Total ARF1 levels in whole cell lysates were assessed by immunoblotting, with actin as a loading control. ( E ) Densitometry analysis of ARF1 activation in WT and Δ sseI STM (MOI = 10) infected HeLa cells relative to uninfected controls. Data represent the mean ± SEM of three independent experiments. ( F ) Immunoblot and densitometry analysis of Active ARF1 protein levels after ARF1-GTP pulldown from isolated peroxisomes, cells infected with WT or Δ sseI STM, or Δ sseI STM complemented with pQE60-SseI clones (ΔGKM, SKL, full length (FL) (MOI = 10). Densitometry analysis represents three independent experiments. ( G ) Fold proliferation of WT or Δ sseI STM (MOI = 10) in HeLa cells transfected with full-length or constitutively active (Q71L) ARF1 or silenced ARF1. Data represent the mean ± SEM of three independent experiments. ( H ) ELISA analysis of PI (4,5) P2 (PIP2) levels on isolated peroxisomes from PEROXO-tag expressing HeLa cells infected with WT or Δ sseI STM. Data from three independent experiments are shown. ( I , J ) Fold proliferation of STM in HeLa cells after silencing PIP4K2A ( I ) or PIP4K2B ( J ). Data represent the mean ± SEM of three independent experiments. ( K ) Fold proliferation of WT STM (MOI = 10) in HeLa cells treated with PIP4K inhibitor THZ-P1-2 (1 µM). Data represent the mean ± SEM of three independent experiments. ( L , M ) Fold proliferation of WT STM (MOI = 10) in human monocyte-derived dendritic cells (Mo-DCs) ( L ) and human monocyte-derived macrophages (MDMs) ( M ) treated with PIP4K inhibitor THZ-P1-2 (1 µM). Data are from four healthy human donors. Data information: Data were analyzed using one-way ANOVA (Sidak’s multiple comparisons test), ( E ) (* p = 0.0211, * p = 0.0138), ( F ) (** p = 0.0092, ** p = 0.0058), ( G ) (**** p < 0.0001), and ( H ) (**** p < 0.0001), ‘ns’ denotes no significant difference. ( I – M ) Data were analyzed using student’s t -test, ( I ) (** p = 0.0091), ( J , K ) (** p = 0.0046), ( L ) (*** p = 0.0002), ( M ) ((*** p = 0.0003). ‘ns’ denotes no significant difference. .

Article Snippet: PIP2 antibody , Novus Biologicals , NBP2-76433.

Techniques: Western Blot, Isolation, Infection, Expressing, Control, In Vitro, Binding Assay, Purification, Staining, Activation Assay, Clone Assay, Transfection, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: EMBO Reports

Article Title: Salmonella Typhimurium effector SseI regulates host peroxisomal dynamics to acquire lysosomal cholesterol

doi: 10.1038/s44319-024-00328-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: PIP2 antibody , Novus Biologicals , NBP2-76433.

Techniques: Derivative Assay, Recombinant, Plasmid Preparation, Synthesized, Control, Sequencing, Cloning, Protease Inhibitor, Software

Journal: Nutrients

Article Title: Cell Membrane Fatty Acids and PIPs Modulate the Etiology of Pancreatic Cancer by Regulating AKT.

doi: 10.3390/nu17010150

Figure Lengend Snippet: Figure 5. Dietary PUFAs modify the PIP2/PIP3 ratio in the membrane affecting AKT signaling. (A) Representative PIP3 IHC images of EK mice pancreata fed normal, ω3-, or ω6-enriched diets (n = 4). (B) PIP3 expression score from 0 to 3+, with 0 as no detectable immunostaining, 1 as 10–30% immunostaining, 2 as 30–60%, and 3 as >60%. The numerical score represents the average of 2 independent investigators. (C) Representative images of PIP3 immunocytochemical staining of PDAC cell lines incubated with DHA or LA at 40 µM for 48 h. PIP3 immunostaining score of (D) Panc-1, (E) MiaPaca-2, and (F) AsPC-1 cells. Staining was scored from 0 to 3+, with 0 as no detectable immunostaining, 1 as 10–30% immunostaining, 2 as 30–60%, and 3 as >60%. (G) Representative images of PIP3 immunocytochemical staining of the Panc-1 cell line incubated with BSA (left image), DHA 40 µM combined with 1 µM of exogenous PIP3 (middle image), and LA 40 µM combined with 1 µM of exogenous PIP2 (n = 3). (H) Western blot images of the Panc-1 pAKT levels after the exogenous supplementation of PIP2 and PIP3 to the PUFAs treatment. Results are expressed as the mean ± standard deviation (SD). Asterisks above a group define significance against the corresponding untreated control: * p < 0.05; ** p < 0.005; *** p < 0.0005. Results are expressed as mean ± standard deviation (SD).

Article Snippet: Primary antibodies PIP3 and PIP2 were purchased from Echelon Biosciences Inc. (Z-P345) and Santa Cruz (sc-53412), respectively, and diluted to 1:100, then 200 μL/well were added to the chamber slides and incubated overnight at 4 ◦C.

Techniques: Membrane, Expressing, Immunostaining, Staining, Incubation, Western Blot, Standard Deviation, Control

Journal: bioRxiv

Article Title: Neuronal growth regulator 1 (NEGR1) promotes synaptic targeting of glutamic acid decarboxylase 65 (GAD65)

doi: 10.1101/2022.02.08.479601

Figure Lengend Snippet: (A, B) CHO cells transfected with pcDNA3 or NEGR1 were either co-transfected with mCherry-D4H to visualize cholesterol (A) or immunolabelled for ganglioside GM4 (B) . Bars, 20 µm. Graphs show ratios of D4H and GM4 labelling levels at the PM and in cytoplasm (mean + SEM, n = 20). *p, Mann Whitney test. (C) Lipid raft markers gangliosides GM4 and GD3 and phosphatidylinositol 4,5-bisphosphate (PIP2) analyzed in triplicate by dot blot in brain homogenates (BH) and synaptosomes (synapt.) from a representative pair of NEGR1+/+ and NEGR1-/- littermates. Graphs show mean + SEM synaptic enrichments of the lipids in NEGR1-/- mice relative to the levels in NEGR1+/+ mice set to 1. n = 5 pairs of NEGR1+/+ and NEGR1-/- mice were analyzed. *p, one sample t test vs +/+. Figure 6 source data. Contains dot blot source data for Figure 6.

Article Snippet: Mouse monoclonal antibodies against PIP2 [2C11] were from Novus Biologicals (NB100-2751, DB: 1:1000).

Techniques: Transfection, MANN-WHITNEY, Dot Blot