Journal: PLoS ONE

Article Title: Identification of Leptospira interrogans Phospholipase C as a Novel Virulence Factor Responsible for Intracellular Free Calcium Ion Elevation during Macrophage Death

doi: 10.1371/journal.pone.0075652

Figure Lengend Snippet: (A). P 2 X 7 protein expression in J774A.1 and THP-1 cells. Lane M: protein marker. Lane P 2 X 7 : immunoblotting of P 2 X 7 protein of J774A.1 or THP-1 cells with P 2 X 7 -IgG. Lane C: blank control. (B). Leptospires in J774A.1 or THP-1 cells viewed under the electron microscope after infection with L. interrogans strain Lai for 30 min. The arrows indicate the leptospires within phagocytotic vesicles in the cytosol of J774A.1 or THP-1 cells. (C). Leptospires in J774A.1 or THP-1 cells viewed under the laser confocal microscope after infection with L. interrogans strain Lai for 30 min. The small red spots correspond to the intracellular leptospires (pointed by the arrows), while the large blue plaques correspond to the cell nucleus. (D). Phosphorylation increase of PI-PLCs in J774A.1 and THP-1 cells during infection with L. interrogans strain Lai for the indicated times, determined by Western Blot assay. The images at “0 h” indicate the immunoblotting results of total PI-PLC-β3 or PI-PLC-γ1, pSer537 in PI-PLC-β3 and pTyr759, pTyr783 or pTyr1217 in PI-PLC-γ1 of the J774A.1 or THP-1 cells before infection.

Article Snippet: Using 1∶500 diluted rabbit-IgG against total PLC-β3 or PLC-γ1, pSer537 in PLC-β3, or pTyr759, pTyr783 or pTyr1217 in PLC-γ1 (Santa Cruz) as the primary antibody, and 1∶3000 diluted HRP-conjugated goat anti-rabbit-IgG (Jackson ImmunoResearch) as the secondary antibody, Western Blot assays were performed to detect the phosphorylation of PLC-β3 and PLC-γ1 in the supernatant samples.

Techniques: Expressing, Marker, Western Blot, Microscopy, Infection

Journal: Clinical Cancer Research

Article Title: Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer

doi: 10.1158/1078-0432.ccr-21-1992

Figure Lengend Snippet: Figure 1. PLCg1 expression in colorectal cancer and its association with cetuximab responses. A, Comparison of PLCG1 mRNA expression levels in RAS WT tumors (n ¼ 273) versus normal colonic mucosa (n ¼ 41) from TCGA-COAD dataset. B, PLCG1 mRNA expression of paired RAS WT tumors, normal samples, and liver metastasis from AMC cohort (n ¼ 13). C, Correlation between the levels of PLCG1 expression [RNA-seq TPM gene expression quantification from the Broad Institute Cancer Cell Line Encyclopedia (CCLE)] and cetuximab resistance (drug sensitivity measurements of GDSC from the Cancer Dependency Map Portal) in colorectal cancer RAS WT cell lines (n ¼ 14). P value corresponds to the Spearman correlation test. Drug sensitivity was measured as AUC which corresponds to the AUC in which values of 0 correspond to complete reduction in cell viability and values of 1 correspond to no reduction in cell viability. Fitted linear regression line and its 95% CIs are indicated by the red line and shaded area, respectively. D, Cetuximab resistance of RAS WT colorectal cancer cell lines grouped by low or high PLCG1 expression based on the analysis of GDSC and CCLE data (n ¼ 14; unpaired t test). E, Cell viability of colorectal cancer cell lines measured 72 hours after initial exposure to cetuximab (n ¼ 4, two-way ANOVA test). F, Western blotting of EGFR and PLCg1 downstream signaling. b-Actin was used as the loading control. G–I, Cell viability (n ¼ 4, two-way ANOVA test), proliferation, and apoptosis rate of parental and PLCG1 overexpressing SW48 cells (pTriex-PLCG1) upon 72 hours of treatment with cetuximab (unpaired t test). J–L, Cell viability (n ¼ 4, two-way ANOVA test), proliferation, and apoptosis rate of shControl and shPLCG1 CACO-2 cell line after 72 hours of treatment with cetuximab (unpaired t test). Results are presented as the mean SEM. (, P ≤0.05; , P ≤0.01; , P ≤0.0001).

Article Snippet: For PLCG1 knockdown, CACO-2 cells were infected with PLCG1 shRNA lentiviral particles and scrambled control (#sc-29452 and #sc-108080; Santa Cruz Biotechnology).

Techniques: Expressing, Comparison, RNA Sequencing, Gene Expression, Western Blot, Control

Journal: Clinical Cancer Research

Article Title: Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer

doi: 10.1158/1078-0432.ccr-21-1992

Figure Lengend Snippet: Figure 3. PLCg1 expression in colorectal cancer tumors and its association with cetuximab responses. A, IHC analysis of PLCg1 expression in human colorectal cancer specimens. Intensity of PLCg1 cytoplasmic staining in tumor cells ranges from 0 (absence of staining) to 3 (maximal intensity; magnification, 200). B–E, Kaplan– Meier curves of PFS and OS of patients with colorectal cancer expressing high and low levels of PLCg1. Median time of survival for both analyses is shown in the picture. P value of the Kaplan–Meier curves was calculated using the log-rank test.

Article Snippet: For PLCG1 knockdown, CACO-2 cells were infected with PLCG1 shRNA lentiviral particles and scrambled control (#sc-29452 and #sc-108080; Santa Cruz Biotechnology).

Techniques: Expressing, Staining

Journal: Clinical Cancer Research

Article Title: Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer

doi: 10.1158/1078-0432.ccr-21-1992

Figure Lengend Snippet: Figure 4. Role of PLCg1 nSH2-CSH2 domains and its interaction with SHP2 for cetuximab resistance. A and B, Western blotting of CACO-2 (shControl and shPLCG1) and SW48 (parental and pTriex-PLCG1 overexpressed) cells treated with cetuximab for 72 hr. EGFR downstream signaling, namely ERK and AKT are shown. b-actin was used as the loading control. C, Cell viability of parental SW48 and PLCG1-overexpressing cells: full length WT; constitutively active mutant - D1019K; constitutively inactive mutant - H335A; deletion of both nSH2-CSH2 tandem domains - DSH2; and deletions of SH3 domain - DSH3 (n ¼ 3). Analysis was performed using two-way ANOVA test (P < 0.0001). D, Western blotting of EGFR downstream signaling of SW48 parental and overexpressing PLCG1 WT and PLCG1 DSH2 mutant, after 72 hr of cetuximab treatment. b-Actin was used as the loading control. E, Co-immunoprecipitation of PLCg1 with anti-Stag antibody and Western blot analysis of PLCg1 and SHP2 of 72 hours cetuximab treated SW48 cells. F, Co-immunoprecipitation of SHP2 with anti-SHP2 antibody and Western blot analysis of PLCg1 and SHP2 of 72 hours cetuximab treated SW48 cells. G, Proximity ligation assay of PLCg1 and SHP2 using anti-Stag and anti-SHP2 antibodies in SW48 parental, PLCG1 WT- and DSH2-overexpressing cells and corresponding quantification (n ¼ 4). H, Cell viability of SW48 parental and overexpressing PLCG1 WT and DSH2 mutant, treated with cetuximab and cetuximab þ SHP099 for 72 hours (n ¼ 3). I, Western blotting of EGFR downstream signaling of SW48 parental and overexpressing PLCG1 WT and DSH2 mutant, treated with cetuximab and cetuximab þ SHP099. Results are presented as the mean SEM. Statistical analysis was performed using unpaired t test [not significant (ns), P > 0.05; , P < 0.01].

Article Snippet: For PLCG1 knockdown, CACO-2 cells were infected with PLCG1 shRNA lentiviral particles and scrambled control (#sc-29452 and #sc-108080; Santa Cruz Biotechnology).

Techniques: Western Blot, Control, Mutagenesis, Immunoprecipitation, Proximity Ligation Assay

Journal: Clinical Cancer Research

Article Title: Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer

doi: 10.1158/1078-0432.ccr-21-1992

Figure Lengend Snippet: Figure 5. Zebrafish xenotransplant model of CACO-2 shControl and SW48-PLCg1 cells treated with cetuximab and SHP099. A, Cetuximab-resistant CACO-2 shControl and SW48-PLCg1 were fluorescently lableled with DiI (red) and injected into the PVS of 2 dpf nacre/casper zebrafish larvae. Zebrafish xenografts were treated in vivo with cetuximab and SHP099 and compared with untreated controls regarding tumor size, cell death, and metastasis. B–E’, At 4 dpi, zebrafish CACO-2 xenografts were imaged on PVS and over the zebrafish body by confocal microscopy. F, Analysis of CACO-2 tumor size (shControl vs. shControl cetuximab þ SHP099, P ¼ 0.0017; shControl cetuximab vs. shControl cetuximab þ SHP099, P ¼ 0.0096). G, Analysis of CACO-2 tumors activated caspase 3 (apoptosis; shControl vs. shControl cetuximab þ SHP099, P < 0.0001; shControl cetuximab vs. shControl cetuximab þ SHP099, P < 0.0001). H–K’, At 4 dpi, zebrafish SW48–PLCg1 xenografts were imaged on PVS and over the zebrafish body by confocal microscopy. L, Analysis of SW48–PLCg1 tumor size (SW48–PLCg1 vs. SW48–PLCg1 cetuximab þ SHP099, P < 0.0001; SW48–PLCg1 cetuximab vs. SW48–PLCg1 cetuximab þ SHP099, P < 0.0001). M, Analysis of SW48–PLCg1 tumors activated caspase 3 (apoptosis; SW48–PLCg1 vs. SW48–PLCg1 cetuximab þ SHP099, P < 0.0001; SW48–PLCg1 cetuximab vs. SW48–PLCg1 cetuximab þ SHP099, P < 0.0001). N, Representative image of micrometastasis in CHT. O, Analysis of CACO-2 metastasis (shControl cetuximab versus shControl cetuximab þ SHP099, P ¼ 0.0219; shControl versus shPLCg1 cetuximab þ SHP099, P ¼ 0,0046). P, Analysis of SW48–PLCg1 metastasis (SW48–PLCg1 cetuximab vs. SW48–PLCg1 cetuximab þ SHP099, P ¼ 0.0127; SW48–PLCg1 vs. SW48–PLCg1 cetuximab þ SHP099, P ¼ 0.0116). The outcomes are expressed as AVG SEM (ns > 0.05; , P ≤0.05; , P ≤0.01; , P ≤0.001; , P ≤0.0001). Scale bars ¼ 50 mm.

Article Snippet: For PLCG1 knockdown, CACO-2 cells were infected with PLCG1 shRNA lentiviral particles and scrambled control (#sc-29452 and #sc-108080; Santa Cruz Biotechnology).

Techniques: Injection, In Vivo, Confocal Microscopy

Journal: Clinical Cancer Research

Article Title: Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer

doi: 10.1158/1078-0432.ccr-21-1992

Figure Lengend Snippet: Figure 6. PLCg1-dependent mechanism of cetuximab resistance. Representative mechanism of cetuximab resistance dependent on PLCg1 expression levels, based on findings described above. In colorectal cancer cells, binding of PLCg1 to SHP2 is expected to transduce intracellular signs downstream of EGFR in parallel to ERK and AKT canonic signaling. When cancer cells are treated with anti-EGFR therapies, EGFR downstream signaling is inhibited. However, PLCg1–SHP2 axis is able to sustain downstream signaling, when levels of PLCg1 are high, inducing cetuximab resistance. Combination of EGFR-targeted therapy with anti-SHP2 inhibitors benefits colorectal cancer tumors with PLCg1 overexpression.

Article Snippet: For PLCG1 knockdown, CACO-2 cells were infected with PLCG1 shRNA lentiviral particles and scrambled control (#sc-29452 and #sc-108080; Santa Cruz Biotechnology).

Techniques: Expressing, Binding Assay, Transduction, Over Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: OSBP-Related Protein 5L Maintains Intracellular IP 3 /Ca 2+ Signaling and Proliferation in T Cells by Facilitating PIP 2 Hydrolysis.

doi: 10.4049/jimmunol.1900671

Figure Lengend Snippet: FIGURE 4. ORP5L maintains Ca2+ signaling. (A) The amount of PIP2 in the PM preparation from T cells transfected with wild-type ORP5L, ORP5L (Mut2), or ORP5L (Mut4). Pan-cadherin (p-cad) is used as internal control in the PM. The quantification of the dot blots indicates relative PIP2 level; the corresponding graph is shown in the right-hand panel. (B) The amount of DAG in the PM (left) and the IP3 production (right) upon anti-CD3 (10 mg/ml) stimulation for 3 min in the indicated cells. (C) Ca2+ efflux in T cells transfected with the indicated constructs, induced by anti-CD3 (10 mg/ml). (D) The amount of PIP2 in the PM preparation from T cells cotransfected with siNT, siPLCg1 or siORP5L plus Mock, ORP5L (Mut2) or ORP5L (Mut4). P-cad is used as internal control in the PM. The quantification of the dot blots indicates relative PIP2 level; the corresponding graph is shown in the right-hand panel. (E) The amount of DAG in the PM (left) and the IP3 production (right) upon anti-CD3 (10 mg/ml) stimulation for 3 min in the indicated cells. (F) Ca2+ efflux in T cells transfected with the indicated constructs, induced by anti-CD3 (10 mg/ml). Data represent mean 6 SD of at least three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test.

Article Snippet: The siPLCg1 was purchased from Santa Cruz Biotechnology (sc-29452).

Techniques: Transfection, Control, Construct