santa cruz sp1  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: Identification of a lipid oxygen radical defense pathway and its epigenetic control

doi: 10.1038/s41467-025-67304-4

Figure Lengend Snippet: a , b Western blot (WB) of ZDHHC20 isoforms (Short-S, 42 kDa; 20 L, ~49, 62, 69 kDa) and GAPDH (loading control) in a, indicated cancer cell lines and SARS-CoV-2-infected Calu-3 cells (MOI 0.1, 24 h); b primary human airway epithelia from donors of different ages, untreated or SARS-CoV-2-infected (MOI 0.1, 48 h). c Overview of the ZDHHC20 locus (first intron, exon, 5′UTR; hg19). Tracks show in-frame start codons (ATGs), transcript (GENCODE), histone modifications (ENCODE: H3K9me3, repression; H3K4me1, enhancer; H3K27Ac, activation; H3K4me3, promoter), and SETDB1/KAP1, ATF2, SP1, FOXA1 ChIP-seq peaks. d WB as in a, total and phospho-ATF2 in HeLa cells (siCtrl or siATF2), untreated or treated with proaerolysin (10 ng/ml, 1 h at 37 °C, incubated further 8 h). Quantification of 20 L isoforms shown as mean ± SEM, dots represent independent experiments ( n = 10); p values by two-way ANOVA, Sidak’s correction. e WB as in a, in U2OS expressing ATF2 (WT, 6D, 6 A), or FOXA1 and SP1. Results mean ± SEM, independent experiments (Control - Ctrl, n = 6; others, n = 3); p values versus Ctrl, two-way ANOVA, Dunnett’s correction. f Cell viability of HeLa cells (ATP assay), normalized to untreated siCtrl (100%), mean ± SD from one of three representative assays ( n = 34 replicates); p values vs. siCtrl, two-way ANOVA, Dunnett’s correction. g Schematic: KAP1-KZFPs recruit SETDB1, promoting H3K9me3-mediated repression and active histone mark removal (adapted from ref. 36). h WB as in d, U2OS cells siCtrl, siSETDB1, or siKAP1-depleted (72 h). Results mean ± SEM, independent experiments ( n = 6); p values vs. Ctrl, two-way ANOVA, Dunnett’s correction. i WB as in ( h ) siSETDB1 cells expressing WT or catalytically inactive SETDB1 mutant (Mut). j WB as in a, in HAP-1 wild-type or KAP1-knockout cells. k mRNA quantification across ZDHHC20 transcripts (coding regions: C1, C2; 5’UTR positions: 1–5) in HAP-1 wild-type or KAP1-knockout cells, mean ± SEM, biologically independent experiments, p values vs. normalized WT, two-way ANOVA, Dunnett’s correction. Source data and entire blots provided as a Source Data file and supplementary information.

Article Snippet: The following human siRNA were purchased from Santa Cruz: SP1 (sc-44221), FOXA1 (sc-37930).

Techniques: Western Blot, Control, Infection, Activation Assay, ChIP-sequencing, Incubation, Expressing, ATP Assay, Mutagenesis, Knock-Out

Journal: Nature Communications

Article Title: Identification of a lipid oxygen radical defense pathway and its epigenetic control

doi: 10.1038/s41467-025-67304-4

Figure Lengend Snippet: a Overview of ZDHHC20 locus (intron, exon, 5′UTR; hg19). Tracks: transcription start codons (ATGs), transcript annotations (GENCODE), ChIP-seq peaks (ENCODE: SETDB1/KAP1, ATF2, SP1, FOXA1), ZNF354A and KZFP peaks from HEK293T cells (squish view,) from KRABopedia ,( https://tronoapps.epfl.ch/web/krabopedia/ ); b Western blot (WB) of ZDHHC20 isoforms (Short-S, 20L) and GAPDH (loading control) in U2OS cells depleted of SETDB1, KAP1, or indicated KZFPs. c Immunoprecipitation (IP) of endogenous KAP1 in U2OS cells overexpressing HA-tagged KZFPs, untreated or treated with proaerolysin toxin (10 ng/ml, 1 h at 37 °C, washed, incubated further 8 h). Blots: co-IP fractions and total cell extracts (TCE). d IP-KAP1 as in ( c ) from siControl (Ctrl) or siATF2 U2OS cells expressing ZNF354A-FLAG complemented with indicated ATF2 constructs (WT, 6D, 6 A; 24 h). Results mean ± SEM; dots represent independent experiments ( n = 3); P values vs. untreated, two-way ANOVA, Tukey’s correction. e WB as (b) in human primary lung epithelial cells infected with SARS-CoV-2 (MOI 0.1). f IP-KAP1 in siKAP1 U2OS cells expressing KAP1-WT or S473A mutant, treated with aerolysin as in ( c ). Blots show KAP1 and phospho-Ser/Thr proteins in IP fractions. g WB of TCEs as (b) in siKAP1 U2OS cells expressing KAP1 WT, S473A (S/A), or S473D (S/D). h IP-ZNF354A from Calu-3 cells infected with SARS-CoV-2 (24 h), aerolysin-treated U2OS cells (as in c), or siCtrl/siZNF354A-depleted U2OS cells. Blots show ZNF354A and phospho-Ser/Thr proteins (IP fractions) and ZNF354A and GAPDH (TCE). i IP-ZNF354A -HA from U2OS cells expressing indicated ZNF354A-FLAG and ATF2 constructs, aerolysin-treated as ( c ). Blots: IP fractions (KAP1, ATF2, phospho-Ser/Thr); TCE (total/pS473-KAP1, ATF2-HA). j Model of ATF2-KAP1-ZNF354A repressive complex before and after stress. Source data and entire blots provided as a Source Data file and supplementary information.

Article Snippet: The following human siRNA were purchased from Santa Cruz: SP1 (sc-44221), FOXA1 (sc-37930).

Techniques: ChIP-sequencing, Western Blot, Control, Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, Expressing, Construct, Infection, Mutagenesis

Journal: bioRxiv

Article Title: VEGFR2 blockade overcomes acquired KRAS G12D inhibitor resistance driven by PI3Kγ activation

doi: 10.1101/2025.09.16.676456

Figure Lengend Snippet: (A) Relative luciferase reporter activity of two VEGFA promoter regions (1-500 bp and 500-1000 bp upstream of the transcription starting site) in parental and resistant cells. pGL basic was a negative control. The graph indicates the normalization of firefly luminescence by Renilla luminescence. All experiments were repeated three times. Data represent the mean□±□SD. **P <□0.01 and ***P <□0.001. (B) Levels of SP1, Lamin B, and α-tubulin in fractionated cytoplasmic and nuclear protein samples of PANC-1-P and PANC-1-R cells. SP1 expression was normalized by Lamin B expression. (C) Levels of SP1, p-VEGFR2, and VEGFR2 of resistant cells that were transfected with negative control (N/C) or SP1 siRNA. The values indicate the normalized band intensity by vinculin. (D) Deleting 5-bp in the predicted SP1 binding site of the VEGFA promoter region significantly decreased the VEGFA mRNA expression. VEGFA transcriptional levels in PANC-1-R cells transfected with wild-type and deletion vectors were plotted. The values are normalized with 18s rRNA. All experiments were performed in triplicate. The graph indicates the mean ± SD. ***P <□0.001. (E) Representative immunofluorescence images for p-VEGFR2 (green) and SP1 (red) in PANC-1-R cells after transfection with wild-type and deletion vectors. Nuclei were stained with DAPI (blue). Scale bar: 65.3 μm.

Article Snippet: Negative control and SP1 siRNA were purchased from Bioneer (Daejeon, Republic of Korea).

Techniques: Luciferase, Activity Assay, Negative Control, Expressing, Transfection, Binding Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: VEGFR2 blockade overcomes acquired KRAS G12D inhibitor resistance driven by PI3Kγ activation

doi: 10.1101/2025.09.16.676456

Figure Lengend Snippet: (A) Levels of PtdIns (3,4,5) P 3 of parental and resistant cells following treatment with VEH or 1 μM MRTX1133. All experiments were repeated three times. Data represent the mean□±□SD. ***P <□0.001. (B) Levels of KRAS, p110α, p110β, p110γ, and vinculin in parental and resistant cell lines. The values indicate the normalized band intensity by vinculin. (C) The KRAS protein in parental and resistant cells is immunoprecipitated, and the p85, p101, p110α, p110β, and p110γ protein levels are analyzed. The values were normalized by KRAS. The graph indicated the normalized value of AGS, SNU-C2B, and PANC-1 parental and resistant cells. **P <□0.01. (D) p110γ proteins in parental and resistant cells were immunoprecipitated, and p101 protein levels were analyzed. The values of p101 were normalized by p110γ. (E) Representative immunofluorescence images for p-VEGFR2 (green) and SP1 (red) in PANC-1 parental and resistant cells after treatment with 1 μM of eganelisib. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Relative MFI value of SP1 and p-VEGFR2. **P <□0.01 and ***P < 0.001. (F) The viability of resistant cells after treatment with MRTX1133 and/or eganelisib. The experiment was repeated four times. Error bars indicate the mean□±□SD. (G) Combination index values were calculated after the combination treatment of 10 μM MRTX1133 and 10 μM eganelisib in resistant cells. All experiments were performed in triplicate. Data represent the mean□±□standard error of the mean (SEM).

Article Snippet: Negative control and SP1 siRNA were purchased from Bioneer (Daejeon, Republic of Korea).

Techniques: Immunoprecipitation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: VEGFR2 blockade overcomes acquired KRAS G12D inhibitor resistance driven by PI3Kγ activation

doi: 10.1101/2025.09.16.676456

Figure Lengend Snippet: (A) The number of migrated MRTX1133-resistant cells treated with VEH and eganelisib. The graph indicates the number of migrated cells relative to those treated with VEH. The experiment was independently performed three times. **P <□0.01. (B) Levels of ZEB1, vimentin, and vinculin in PANC-1-P and PANC-1-R cells treated with 1 μM eganelisib. The values represent the normalized band intensities by vinculin. (C) The number of migrated MRTX1133-resistant cells transfected with siRNA for SP1 relative to those transfected with the negative control siRNA. The experiment was independently performed three times. **P <□0.01. (D) Levels of SP1, ZEB1, vimentin, and vinculin in PANC-1-R cells after transfection with siRNA for negative control or SP1. The value indicates the normalized values by vinculin intensity. (E) The number of migrated KDR -KO resistant cells by TLCV2-sg KDR after treatment with doxycycline relative to those treated with VEH. The experiment was independently performed three times. *P <□0.05 and **P <□0.01. (F) The number of migrated parental and resistant cells after treatment with 50 μg/mL ramucirumab. The experiment was independently performed three times. **P <□0.01 and ***P <□0.001.

Article Snippet: Negative control and SP1 siRNA were purchased from Bioneer (Daejeon, Republic of Korea).

Techniques: Transfection, Negative Control