pi4kiia  (Proteintech)

Journal: bioRxiv

Article Title: Lipid Transfer Proteins and PI4KIIα Generate a Phosphoinositide-Linked Proteome

doi: 10.64898/2025.12.17.694959

Figure Lengend Snippet: a-b , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against PI4KIIα. After 24h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for IF staining against PI4KIIα and PI4,5P 2 . PI4,5P 2 levels were quantified using ImageJ ( d ). n=3, 15 cells from each independent experiment. c , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against both PITPα and PITPβ or PI4KIIα. After 24h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for WB. n=3 independent experiments. d-e , MDA-MB-231 Cas cells with PITPβ KO and control non-targeted KO were transfected with control siRNAs or siRNAs against PI4KIIα, PITPα or both PI4KIIα and PITPα. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for WB against PI4,5P 2 ( a ), PI4P ( b ), and PI3,4,5P 3 ( b ). n=3 independent experiments. For all graphs, data are presented as the mean ± SD. Scale bar, 5 µm

Article Snippet: The monoclonal antibodies that were used are against p53 (clone DO-1, #SC-126, Santa Cruz Biotechnology), p53 (clone 7F5, #2527, Cell Signaling), pAkt S473 (clone 193H12, #4058, Cell Signaling), HA-tag (clone C29F4, #3724, Cell Signaling), GAPDH (clone 0411, #sc-47724, Santa Cruz Biotechnology), β-actin (clone 13E5, #4970, Cell Signaling), Lamin B2 (clone D8P3U, #12255, Cell Signaling), IQGAP1 (clone D-3, #sc-374307, Santa Cruz Biotechnology), Talin-1 (clone C-9, #sc-365875, Santa Cruz Biotechnology), Ku80 (clone C48E7, #2180, Cell Signaling), and polyclonal antibodies against PITPα (#16613-1-AP, ThermoFisher), PITPβ (#ab127563, abcam), PITPNC1 (IF, #ab222078, abcam), PITPNC1 (WB, #NBP2-19842, Novus), PI4KIIα (#NBP2-44158, Novus).

Techniques: Transfection, Control, Staining

Journal: bioRxiv

Article Title: Control of antigen presentation on MHC-I by a bacterial secretion system

doi: 10.1101/2025.11.19.689332

Figure Lengend Snippet: (A-B) Primary hMDMs infected with wild-type or eccCa1::Tn Mtb at MOI 2.5 for 72 hours, treated with prazosin, gefitinib, or DMSO alone for 2 hours, and stained for P62. A) Representative fluorescence confocal microscope images. White arrows indicate intracellular bacteria colocalized with P62. B) Relative quantification of colocalization between intracellular Mtb and P62, as measured by analysis of local fluorescence intensity correlation (see Methods) for n = 3 donors (* p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). (C-D) ATG7 knockout THP-1 macrophages or the parental wild-type line were infected with Mtb at MOI 2.5 and harvested for MHC-I peptide quantification by SureQuant 24 hours post-infection. C) Heatmap of relative abundances of MHC-I peptides (mean of n = 3 replicate experiments). D) Relative abundance of EsxA 28-36 and EsxGS 3-11 presented on MHC-I for n = 3 replicate experiments. (E-F) Primary hMDMs infected with wild-type or eccCa1::Tn Mtb at MOI 2.5 for 72 hours, treated with prazosin, gefitinib, or DMSO alone for 2 hours, and stained for ORP9. E) Representative fluorescence confocal microscope images. White arrows indicate intracellular bacteria colocalized with ORP9. F) Relative quantification of colocalization between intracellular Mtb and ORP9, as measured by analysis of local fluorescence intensity correlation (see Methods) for n = 3 donors (* p < 0.05, ** p < 0.01, one-way ANOVA with Tukey’s multiple comparisons test). (G-H) PI4K2A knockout THP-1 macrophages or the parental wild-type line were infected with Mtb at MOI 2.5 and harvested for MHC-I peptide quantification by SureQuant 24 hours post-infection. G) Heatmap of relative abundances of MHC-I peptides (mean of n = 3 replicate experiments). H) Relative abundance of EsxA 28-36 and EsxGS 3-11 presented on MHC-I for n = 3 replicate experiments (* p < 0.05, one-way paired ANOVA with Dunnett’s multiple comparisons test). (I-J) MPEG1 knockout THP-1 macrophages or the parental wild-type line were infected with Mtb at MOI 2.5 and harvested for MHC-I peptide quantification by SureQuant 24 hours post-infection. I) Heatmap of relative abundances of MHC-I peptides (mean of n = 3 replicate experiments). J) Relative abundance of EsxA 28-36 and EsxGS 3-11 presented on MHC-I for n = 3 replicate experiments.

Article Snippet: Membranes were blocked for 1 hr at room temperature with blocking buffer (Licor) and incubated overnight at 4 °C with anti-PI4K2A antibody (Santa Cruz Biotechnology, sc-390026), diluted 1:1000 in antibody diluent (Licor).

Techniques: Infection, Staining, Fluorescence, Microscopy, Bacteria, Quantitative Proteomics, Knock-Out

Journal: bioRxiv

Article Title: Astrocytes mobilize a broader repertoire of lysosomal repair mechanisms than neurons

doi: 10.1101/2025.09.07.674666

Figure Lengend Snippet: (a) Cocultured hippocampal astrocytes and neurons treated with 1.5 mM LLOMe or equivalent volume of DMSO solvent control for the time indicated and immunostained for GFP (LC3), PI4K2A and LAMP1. Note: insets showing astrocyte PI4K2A with LLOMe treatment is enhanced to show PI4K2A rings in astrocytes. (b and c) Representative line scans of PI4K2A and LAMP1 puncta in astrocytes and neurons after 30 (b) and 60 (c) minutes of LLOMe. Overlapping peaks of PI4K2A and LAMP1 are denoted with paired green and magenta dots. (d and e) Zoom-ins of Hoechst/PI4K2A/LAMP1 merged images in LLOMe or DMSO treated astrocytes (d) and neurons (e) . Yellow arrowheads indicate PI4K2A puncta that are copositive for LAMP1 puncta. (f) Cocultured hippocampal astrocytes and neurons treated with 1.5 mM LLOMe or equivalent volume of DMSO solvent control for the time indicated and immunostained for GFP (LC3), ORP9 and LAMP. (g and h) Representative line scans of ORP9 and LAMP1 puncta in astrocytes and neurons after 30 (g) and 60 (h) minutes of LLOMe. Overlapping peaks of ORP9 and LAMP1 peaks are denoted by paired green and magenta dots. (i and j) Corresponding quantification of total ORP9 puncta area normalized to soma area in LLOMe or DMSO treated astrocytes (i) and neurons (j) . Horizontal bars represent the means of the biological replicates ± SEM. Shown are p-values from a LME model with Holm’s post hoc correction; N=33-36 astrocytes (i) and N=33-36 neurons (j) from 3 independent experiments. (k and l) Corresponding quantification of total LAMP1 puncta area copositive for ORP9 in LLOMe or DMSO treated astrocytes (k) and neurons (l) . Horizontal bars represent the means of the biological replicates ± SEM. Shown are p-values from a LME model with Holm’s post hoc correction; N=28-32 astrocytes (k) and N=28-31 neurons (l) from 3 independent experiments. Throughout the figure, neurons are outlined in orange and astrocytes are outlined in blue. Scale bars, 10 µm.

Article Snippet: Primary antibodies for immunofluorescence include antibodies against GFP (Aves Lab Inc., GFP-1020), MAP2 (Millipore Sigma, ab5622), β3 tubulin (R&D Systems, MAB1195), EEAT1/GLAST (abcam, ab416), S100β (Sigma, S2532), LAMP1 (abcam, ab25245), TAX1BP1 (abcam, ab176572), p62 (abcam, ab109012), CHMP2B (abcam, ab33174), ALIX (Biolegend, 634502), TBC1D15 (abcam, ab121396), IST1 (Proteintech, 19842-1-AP), CHMP2A (Proteintech, 10477-1-AP), PI4K2A (A kind gift of Dr. Pietro De Camilli, Yale University, New Haven, CT) , and ORP9 (Santa Cruz, sc-398961).

Techniques: Solvent, Control

Journal: bioRxiv

Article Title: Astrocytes mobilize a broader repertoire of lysosomal repair mechanisms than neurons

doi: 10.1101/2025.09.07.674666

Figure Lengend Snippet: Data extracted from the RNA sequencing database generated by the lab of Dr. Ben Barres showing the mRNA expression profiles of key lysosomal repair proteins in mouse cortical neurons and astrocytes ( brainrnaseq.org ). (a-d) mRNA levels of ESCRT proteins, Chmp2b (a) , Chmp2a (b) , Pdcd6ip (ALIX) (c) , and Pdcd6 (ALG-2) (d) in astrocytes and neurons. (e) mRNA levels of Tbc1d15 in astrocytes and neurons. (f and g) mRNA levels of PITT pathway proteins, Pi4k2a (f) and Osbpl9 (ORP9) (g) in astrocytes and neurons. (h) mRNA levels of Lgals3 (Galectin-3), Lgals8 (Galectin-8) and Lgals9 (Galectin-9) in astrocytes and neurons.

Article Snippet: Primary antibodies for immunofluorescence include antibodies against GFP (Aves Lab Inc., GFP-1020), MAP2 (Millipore Sigma, ab5622), β3 tubulin (R&D Systems, MAB1195), EEAT1/GLAST (abcam, ab416), S100β (Sigma, S2532), LAMP1 (abcam, ab25245), TAX1BP1 (abcam, ab176572), p62 (abcam, ab109012), CHMP2B (abcam, ab33174), ALIX (Biolegend, 634502), TBC1D15 (abcam, ab121396), IST1 (Proteintech, 19842-1-AP), CHMP2A (Proteintech, 10477-1-AP), PI4K2A (A kind gift of Dr. Pietro De Camilli, Yale University, New Haven, CT) , and ORP9 (Santa Cruz, sc-398961).

Techniques: RNA Sequencing, Generated, Expressing

Journal: bioRxiv

Article Title: Senescent cell survival relies on upregulation of lysosomal quality control mechanisms

doi: 10.1101/2025.03.31.646397

Figure Lengend Snippet: (A) Proliferative and senescent cells were fixed and immunostained for antibodies against PI4K2A (green) and LAMP1 (magenta), scale bar 10 μm. Colocalisation between PI4K2A and LAMP1 (B) was analysed by Pearson’s correlation. Error bars represent SEM; n = 5 independent experiments with at least 6 fields of view per experimental repeat. Student’s t test (unpaired). (C) Proliferative and senescent cells were incubated with 50 μg/ml cycloheximide (CHX) for the timepoints indicated. The abundance of PI4K2A and the indicated proteins were assessed by western blot. (D) mRNA levels of senescent cells, normalised to PUM1 and presented relative to proliferating cells (dashed line) n = 3 independent experimental repeats for PI4K2A and ULK3 and n = 2 independent experimental repeats for CTSB and TFE3 . (E) Representative immunofluorescence image of proliferative and senescent cells expressing the PI(4)P sensor GFP-P4M-SidMx2 immunostained for an antibody against LAMP2 (magenta), scale bar 10 μm. (F) Colocalisation between GFP-P4M-SidMx2 and LAMP2 was analysed by Pearson’s correlation. Error bars represent SEM; n = 3 independent experiments with at least 32 cells per experimental repeat. Student’s t test (unpaired). (G) Representative movie stills of live-cell imaging experiment showing LysoTracker-labelled lysosomes (magenta) in proliferating and senescent cells expressing the PI(4)P sensor GFP-P4M-SidMx2. Scale bar, 10 μm (H) The speed of PI(4)-positive and -negative LysoTracker-labelled lysosomes (μm/frame) are displayed. The mean ± SEM speed from 6 movies per condition are compared by two-way ANOVA followed by Šídák’s multiple comparison. (I) PI(4)P-positive lysosomes in senescent cells display higher MagicRed Cathepsin B activity compared to PI(4)P-positive lysosomes in proliferating cells. Representative movie stills of live-cell imaging experiment showing MagicRed Cathepsin B-labelled lysosomes (red) in proliferating and senescent cells expressing the PI(4)P sensor GFP-P4M-SidMx2. Scale bar, 10 μm. (J) Quantification of PI(4)P-positive and -negative MagicRed Cathepsin B activity in proliferating or senescent cells. Quantification represents mean ± SEM; n = 2 with ≥ 5 movies analysed per condition; two-way ANOVA followed by Šídák’s multiple comparison. *, P < 0.05; **, P < 0.01; ***, P < 0.001;

Article Snippet: The following primary antibodies were used: mouse monoclonal antibodies raised against Lamp1 (H4A3, DSHB, 1:2000), PI4K2A (sc-390026, Santa Cruz, 1:2000), β-Actin (8H10D10, 3700, Cell Signalling Technology, 1:5000), GAPDH (D4C6R, 97166, Cell Signalling Technology, 1:2000), integrin beta 1/CD29 (610467, BD Biosciences, 1:2000), transferrin receptor/CD71 (3B8 2A1, sc-32272, Santa Cruz, 1:1000), GFP (7.1/13.1, Roche, 11814460001, 1:2000), rabbit monoclonal antibodies raised against cathepsin B (D1C7Y, 31718, Cell Signalling Technology, 1:2000), CI-MPR (EPR6599, 124767, Abcam, 1:2000), Lamp1 (D2D11 9091, Cell Signalling Technology, 1:2000), phospho-S6 Ser235/236 (4856, Cell Signaling Technology, 1:2000), S6 (2217, Cell Signaling Technology, 1:1000), phospho-ULK1 Ser757 (D7O6U, 14202, Cell Signaling Technology, 1:1000), ULK1 (D8H5, 8054, Cell Signaling Technology, 1:1000), integrin alpha 5 (EPR7854, ab150361, Abcam, 1:2000), rabbit polyclonal antibodies raised against PI4K2A (15318-1-AP, Proteintech, 1:1000), LDLR (10785-1-AP, Proteintech, 1:2000), CD36 (18836-1-AP, Proteintech, 1:1000), VAPA (15275-1-AP, Proteintech, 1:2000).

Techniques: Incubation, Western Blot, Immunofluorescence, Expressing, Live Cell Imaging, Comparison, Activity Assay