Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a , Domains of PITP class I and II protein family members. PITD, PI transfer domain; FFAT, two phenylalanines in an acidic tract; DDHD, Asp-Asp-His-Asp motif; LNS2, Lipin/Nde1/Smp2 domain. b-c , Confocal images of IF staining against PITPα/PITPβ/PITPNC1 and PI4,5P 2 in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclei were counterstained by DAPI. The nuclear levels of PITPs and PI4,5P 2 were quantified by ImageJ ( c ). n=3, 10 cells from each independent experiment. See expanded images in . d , Quantification of nuclear PITPα/PITPβ levels by IF staining in wild-type and mutant p53 expressing cells, including MCF10A (WT), A549 (WT), HCT116 (WT), BT-549 (R249S), Cal33 (R175H), HS578T (V157F), SUM159 (R158InL), and SUM1315 (C135F). n=3, 10 cells from each independent experiment. See IF images in . e-f , MDA-MB-231 cells were treated with 30 µM cisplatin, 20 µM erastin, 100 µM tBHQ, 100 µM etoposide, 100 µM hydroxyurea, or vehicle for 24 h. The cells were then processed for IF staining against PITPα or PITPβ and PI4,5P 2 . The nuclei were counterstained by DAPI. The nuclear levels of PITPs and PI4,5P 2 were quantified by ImageJ ( f ). n=3, 10 cells from each independent experiment. See expanded images in . g , Confocal IF of PITPα or PITPβ overlaid with the nuclear envelope marker Lamin A/C in MCF10A, A549, MDA-MB-231, SUM1315, and BT-549 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclei were counterstained by DAPI. For all panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar, 5 µm.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Mutagenesis, Expressing, Marker

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a , Confocal images of IF staining against PITPα/PITPβ/PITPNC1 and PI4,5P 2 in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclei were counterstained by DAPI. See quantification in . b-c , 3D sections of IF staining against PITPα in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclei were counterstained by DAPI. Each frame of the 3D sections was over a 0.2 μm thickness. d-e , 3D sections of IF staining against PITPβ in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclei were counterstained by DAPI. Each frame of the 3D sections was over a 0.2 μm thickness. For all panels, Scale bar, 5 µm.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Staining

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a , Confocal images of the IF staining against PITPα/β in wild-type and mutant p53 expressing cells, including MCF10A (WT), A549 (WT), HCT116 (WT), BT-549 (R249S), Cal33 (R175H), HS578T (V157F), SUM159 (R158InL), and SUM1315 (C135F). The nuclei were counterstained by DAPI. See the quantification of the nuclear levels of PITPα/PITPβ in . b , MDA-MB-231 cells treated with 30 µM cisplatin, 20 µM erastin, 100 µM tBHQ, 100 µM etoposide, 100 µM hydroxyurea, or vehicle for 24 h. The cells were processed for IF staining against PITPα and PI4,5P 2 . The nuclei were counterstained by DAPI. See the quantification of nuclear PITPα, PITPβ and PI4,5P 2 in . c, Confocal IF of PITPNC1 overlaid with the nuclear envelope marker Lamin A/C in MDAMB-231 and A549 cells treated with vehicle or 30 μM cisplatin for 24 h. The nuclei were counterstained by DAPI. For all panels, Scale bar, 5 µm.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Staining, Mutagenesis, Expressing, Marker

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a-d, Confocal IF images of PITPα/β overlaid with the nuclear envelope marker Lamin A/C in wild-type and mutant p53 expressing cancer cells, including HCT116 (WT, a ), Cal33 (R175H, b ), HS578T (V157F, c ), and SUM159 (R158InL, d ). The nuclei were counterstained by DAPI. e , MCF10A and SUM1315 cells with the indicated p53 status were transfected with control siRNAs or siRNAs against both PITPα and PITPβ. After 24 h, cells were treated with 30 μM cisplatin or vehicle for 24 h before being processed for IF staining against PI4,5P 2 (red) and the shared epitope of PITPα and PITPβ (green). The nuclei were counterstained by DAPI. See KD validation in and quantification in . n=3, 10 cells from each independent experiment. For all panels, Scale bar, 5 μm.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Marker, Mutagenesis, Expressing, Transfection, Control, Staining, Biomarker Discovery

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a-b , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against PITPα, PITPβ, PITPNC1, or both PITPα and PITPβ. After 24h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for IF staining. The nuclear PI4,5P 2 level was quantified by ImageJ ( b ). n=3, 10 cells from each independent experiment. See KD validation in . c-d , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against both PITPα and PITPβ. After 24 h, cells were treated with 30 μM cisplatin or vehicle for 24 h before being processed for IF staining against PI4P/PI3,4,5P 3 (red) and the shared epitope of PITPα and PITPβ (green). The nuclei were counterstained by DAPI. The nuclear PI4P and PI3,4,5P3 levels were quantified by ImageJ ( d ). n=3, 10 cells from each independent experiment. See KD validation in . e-g , A549, BT-549, MCF10A, and SUM1315 cells were transfected with control siRNAs or siRNAs against both PITPα and PITPβ. After 24 h, cells were treated with 30 μM cisplatin or vehicle for 24 h before being processed for IF staining against PI4,5P 2 (red) and the shared epitope of PITPα and PITPβ (green). The nuclei were counterstained by DAPI. The nuclear PI4,5P 2 levels were quantified by ImageJ ( g ). n=3, 10 cells from each independent experiment. See KD validation in and expanded images in . h , Models showing stress-induced nuclear translocation of PITPα/β to regulate nuclear PIP n puncta. For all panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar, 5 µm.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Staining, Biomarker Discovery, Translocation Assay

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a-b, PLA of p53-PITPα/PITPβ/PITPNC1/PITPNM1/PITPNM2 in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclear p53-PITP foci were quantified ( b ). n=3, 10 cells from each independent experiment. See PLA of p53-PITPNM1/PITPNM2 images in . c, PLA of p53-PITPα/PITPβ/PITPNC1 overlaid with the nuclear envelope marker Lamin A/C in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclei were counterstained by DAPI. d-g , Co-IP of PITPα/PITPβ/Hsp27 with p53 from MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. PITPα IP-ed by p53 ( e ), PITPβ IP-ed by p53 ( f ), and Hsp27 IP-ed by p53 ( g ), as well as PITPNC1/PITPNM1/PITPNM2 were analyzed by WB. n=3 independent experiments. h-k , Co-IP of p53/Hsp27/ αB-crystallin (αBC) with the shared epitope of PITPα/β from MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. p53 IP-ed by PITPα/β ( i ), Hsp27 IP-ed by PITPα/β ( j ), and αBC IP-ed by PITPα/β ( k ) were analyzed by WB. n=3 independent experiments. l-o , In vitro binding of recombinant p53 with PITPα or PITPβ. p53 immobilized on anti-p53 agarose was incubated with PITPα or PITPβ protein. p53 was IP’ed and p53-bound PITPα ( l , m ) or PITPβ ( n , o ) was analyzed by WB and quantified by ImageJ. n=3 independent experiments. p , Model showing stress-induced formation of nuclear PITPα/β, p53, and small heat shock protein (sHSP) complexes. For all panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar, 5 µm.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Marker, Co-Immunoprecipitation Assay, In Vitro, Binding Assay, Recombinant, Incubation

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a, PLA of p53-PITPNM1/PITPNM2 in MDA-MB-231 cells treated with vehicle or 30 µM cisplatin for 24 h. See quantification in . b-c, PLA of p53-PITPα/PITPβ/PITPNC1/PITPNM1/PITPNM2 in A549 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclear p53-PITP PLA foci were quantified by ImageJ ( c ). n=3, 10 cells from each independent experiment. d, PLA of p53-PITPα/PITPβ/PITPNC1/PITPNM1/PITPNM2 overlaid with the nuclear envelope marker Lamin A/C in A549 cells treated with vehicle or 30 µM cisplatin for 24 h. The nuclei were counterstained by DAPI. e, PLA of p53-PITPNM1/PITPNM2 overlaid with the nuclear envelope marker Lamin A/C in MDA-MB-231 treated with vehicle or 30 µM cisplatin for 24 h. The nuclei were counterstained by DAPI. f , 3D sections of p53-PITPβ PLA foci overlaid with Lamin A/C in MDA-MB-231 treated with vehicle or 30 µM cisplatin for 24 h. The nuclei were counterstained by DAPI. Each frame of the 3D sections was over a 0.2 µm thickness. For all panels, data are represented as mean ± SD, p value denotes two-sided paired t-test. Scale bar, 5 µm unless otherwise indicated.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Marker

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a-d, Co-IP of p53/Hsp27/αB-crystallin (αBC) with the shared epitope of PITPα/β in MCF10A cells treated with vehicle or 30 µM cisplatin for 24 h. p53 IP-ed by PITPα/β ( b ), Hsp27 IP-ed by PITPα/β ( c ), and αBC IP-ed by PITPα/β ( d ) were analyzed by WB. n=3 independent experiments. e-f, Co-IP of p53 with the shared epitope of PITPα/β in A549 cells treated with vehicle or 30 µM cisplatin for 24 h. p53 IP-ed by PITPα/β was analyzed by WB ( f ). n=3 independent experiments. g-h, Co-IP of p53 with the shared epitope of PITPα/β in HCT116 cells treated with vehicle or 30 µM cisplatin for 24 h. p53 IP-ed by PITPα/β was analyzed by WB ( h ). n=3 independent experiments. i-n, Co-IP of PITPα/PITPβ with p53 in BT549, Cal33, HS578T, SUM159, SUM1315 cells treated with vehicle or 30 µM cisplatin for 24 h. PITPα/PITPβ IP-ed by p53 ( n ) as well as PITPNC1/PITPNM1/PITPNM2 were analyzed by WB. n=3 independent experiments. For all panels, data are represented as mean ± SD, p value denotes two-sided paired t-test.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a-c, MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PITPα, PITPβ, PITPNC1, or both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for PLA to detect p53-PI4,5P 2 /-PI3,4,5P 3 complexes ( b,c ). n=3, 10 cells from each independent experiment. d-f, A549 cells were transfected with control siRNAs or siRNAs against PITPα, PITPβ, or both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for proximity ligation assay (PLA) to detect p53-PI4,5P 2 /PI3,4,5P 3 complexes ( e,f ). n=3, 10 cells from each independent experiment. g-j , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against PITPα, PITPβ, or both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for IP against p53. p53-bound PI4P/PI4,5P 2 /PI3,4,5P 3 were examined by fluorescent WB and quantified by ImageJ ( h-j ). n=3 independent experiments. See KD validation in . k-l , A549 cells were transfected with control siRNAs or siRNAs against PITPα, PITPβ, or both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for IP against p53. The PI4,5P 2 level on IP-ed p53 ( l ) was quantified by ImageJ. n=3 independent experiments. See KD validation in . For all graphs, data are presented as the mean ± SD, and p values are from two-sided paired t-tests. Scale bar, 5 µm.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Proximity Ligation Assay, Biomarker Discovery

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PITPα, PITPβ, PITPNC1, or both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for WB to validate the KD. The p53 level was quantified by ImageJ. n=3 independent experiments. b , A549 cells were transfected with control siRNAs or siRNAs against PITPα, PITPβ, or both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for WB. The p53 level and protein-bound PI4,5P 2 level were quantified by ImageJ. n=3 independent experiments. c-d, MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PITPα, PITPβ, PITPNC1, or both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for triple fluorescent WB for detecting p53 (Red), GAPDH (Blue), and protein-bound PI4,5P 2 (Green) simultaneously. The protein-bound PI4,5P 2 levels were quantified by ImageJ ( d ). See KD validation in . n=3 independent experiments. e-f, A549 cells were transfected with control siRNAs or siRNAs against PITPα or PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for WB. The protein-bound PIP n levels were quantified by ImageJ ( f ). n=3 independent experiments. g , Model showing the nuclear p53-PIP n signalosome downstream of PITPα/β. For all graphs, data are presented as the mean ± SD, and p values are from two-sided paired t-tests.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Biomarker Discovery

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a-b , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against PITPα, PITPβ, PITPNC1, or both PITPα and PITPβ. After 24h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for IF staining against pAkt S473 . The nuclear pAkt S473 levels were quantified by ImageJ ( b ). n=3, 10 cells from each independent experiment. Scale bar, 5 µm. c-e , BT-549, SUM159, and SUM1315 cells with the indicated p53 status were transfected with control siRNAs or siRNAs against both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for IF staining against PI4,5P 2 . The nuclei were counterstained by DAPI. The nuclear PI4,5P 2 levels were quantified ( d ). n=3, 10 cells from each independent experiment. Scale bar, 5 µm. The double KD of PITPα and PITPβ in SUM159 cells was validated by WB ( e ). See KD validation for BT-549 and SUM1315 cells in . f-g , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PITPα, PITPβ, PITPNC1, or both PITPα and PITPβ. After 24h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for Crystal Violet viability assay. The cells were imaged by EVOS M5000 microscope ( f ) and quantified based on the extracted dye using a plate reader ( g ). Scale bar, 300 µm. h , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against p53, PITPα, PITPβ, PITPNC1, or both PITPα and PITPβ. After 24h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for caspase 3 activity assay. n=3 independent experiments. For all graphs, data are presented as the mean ± SD, and p values are from two-sided paired t-tests.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Staining, Biomarker Discovery, Viability Assay, Microscopy, Caspase-3 Activity Assay

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a-g , BT-549, SUM159, and SUM1315 cells were transfected with control siRNAs or siRNAs against p53, PITPα, PITPβ, or both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for Crystal Violet viability assay. The cells were imaged by EVOS M5000 microscope ( a ) and quantified based on the extracted dye using a plate reader ( b-d ). The KD was validated by WB ( e-g ). n=3 independent experiments. h-j , BT-549, SUM159, and SUM1315 cells were transfected with control siRNAs or siRNAs against p53, PITPα, PITPβ, or both PITPα and PITPβ. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for caspase 3 activity assay. n=3 independent experiments. For all graphs, data are presented as the mean ± SD, and p values are from two-sided paired t-tests. Scale bar, 300 µm.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Viability Assay, Microscopy, Caspase-3 Activity Assay

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a , HEK293FT cells were co-transfected with either HA-tagged wild-type PITPα or PI binding-defective mutant T59D PITPα together with Flag-tagged p53 wt . After 48 h, cells were processed for IP against Flag-tag and analyzed by WB. n=3 independent experiments. b-e , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against the 3’UTR of both PITPα and PITPβ for 24 h. Then the cells were transfected with either HA-tagged wild-type PITPα/β or PI binding-defective mutant T59D PITPα. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for WB ( b ) and IF staining against HA-tag with PI4,5P 2 or pAkt S473 ( c ). The nuclear PI4,5P 2 and pAkt S473 levels in HA-positive cells were quantified by ImageJ ( d, e ). n=3, 10 cells from each independent experiment. Scale bar, 5 µm. f-i , Pearson’s correlation coefficient (Pearson’s r) quantified the correlations between nuclear PI4,5P 2 or pAkt S473 with HA-PITPα wt/T59D levels. n=100 cells pooled from 3 independent experiments. j-k , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against the 3’UTR of both PITPα and PITPβ for 24 h. The cells were then transfected with either HA-tagged wild-type PITPα or PI binding-defective mutant T59D PITPα. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for Crystal Violet viability assay. The cells were imaged by EVOS M5000 microscope ( j ) and quantified based on the extracted dye using a plate reader ( k ). n=3 independent experiments. Scale bar, 300 µm. l-m , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against the 3’UTR of both PITPα and PITPβ for 24 h. The cells were then transfected with either HA-tagged wild-type PITPα or PI binding-defective mutant T59D PITPα. After 24 h, cells were serum starved for an additional 24 h and then scored for invasion through Laminin-coated transwell inserts with 8 µm pores using 10 ng/ml EGF as a chemoattractant for 16 h. The invading cells at the insert bottom were stained with Crystal Violet, imaged ( l ), and quantified ( m ) based on the extracted dye using a plate reader. n=3 independent experiments. Scale bar, 300 µm. n , A model of the stress-induced nuclear accumulation of PITPs and PITP-dependent initiation of the p53-PI signalosome which promotes cell proliferation, survival, and invasion. For all graphs, data are presented as the mean ± SD, and p values are from two-sided paired t-tests.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Binding Assay, Mutagenesis, FLAG-tag, Control, Staining, Viability Assay, Microscopy

Journal: bioRxiv

Article Title: Lipid transfer proteins initiate nuclear phosphoinositide signaling

doi: 10.1101/2023.05.08.539894

Figure Lengend Snippet: a , HEK293FT cells were co-transfected with either HA-tagged wild-type PITPα or PI binding-defective mutant T59D PITPα together with Flag-tagged mutant p53 R175H . After 48 h, cells were processed for IP against Flag-tag and analyzed by WB. n=3 independent experiments. b , HEK293FT cells were co-transfected with either HA-tagged wild-type PITPβ or PI binding-defective mutant T59D PITPα together with Flag-tagged p53 wt . After 48 h, cells were processed for IP against Flag-tag and analyzed by WB. n=3 independent experiments. c , HEK293FT cells were co-transfected with either HA-tagged wild-type PITPβ or PI binding-defective mutant T59D PITPα together with Flag-tagged mutant p53 R175H . After 48 h, cells were processed for IP against Flag-tag and analyzed by WB. n=3 independent experiments. d , MDA-MB-231 cells were transfected with control siRNAs or siRNAs against the 3’UTR of both PITPα and PITPβ for 24 h. The cells were then transfected with either HA-tagged wild-type PITPα or PI binding-defective mutant T59D PITPα. After 24 h, cells were treated with 30 µM cisplatin or vehicle for 24 h before being processed for caspase 3 activity assay. n=3 independent experiments. For all graphs, data are presented as the mean ± SD, and p values are from two-sided paired t-tests. Scale bar, 300 µm.

Article Snippet: HRP-conjugated p53 (#sc-126HRP), PITPα (#sc-13569HRP), PITPβ (#sc-390500HRP) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Binding Assay, Mutagenesis, FLAG-tag, Control, Caspase-3 Activity Assay