Journal: Cancers

Article Title: Short-Term TERT Inhibition Impairs Cellular Proliferation via a Telomere Length-Independent Mechanism and Can Be Exploited as a Potential Anticancer Approach

doi: 10.3390/cancers15102673

Figure Lengend Snippet: Telomerase reverse transcriptase (TERT) inhibition downregulated MYC proto-oncogene bHLH transcription factor (MYC) levels. ( A , B ) Cells were treated with BIBR at indicated concentrations or with DMSO as a control for 24 h. Levels of relative mRNA expression for MYC and TERT in 4134/Late ( A ) and BL41 ( B ) cells are shown. Data represent the mean and SD (bar) from three separate experiments. ( C , D ) Cells treated with BIBR at indicated concentrations or DMSO as a control for 24 h were processed to obtain cytoplasmic and nuclear extracts. Representative Western blots showing cytoplasmic and nuclear protein levels of TERT, MYC, TRF2, and α-tubulin in 4134/Late ( C ) and BL41 ( D ) cells are shown. α-tubulin and TRF2 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. The original Western blots are shown in . Graphs next to the blots show the values in arbitrary units of densitometric analysis performed with ImageJ software. Data represent the mean and SD (bar) from three separate experiments. A significant difference between values in BIBR-treated vs. DMSO-treated cells is shown: * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.

Article Snippet: The expression of TERT, MYC, p65, phosphorylated p65 (p-p65), cyclin-dependent kinase inhibitor 1A (CDKN1A, P21), telomeric repeat binding factor 2 (TRF2), and α-tubulin were evaluated by the following antibodies: TERT (ab32020, Abcam, Cambridge, UK; abx120550, Abexxa, Arlington, TX, USA), NF-κB p65 (ab32536, Abcam), phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), MYC (ab32072, Abcam), P21 (ab109520, Abcam), TRF2 (NB110-57130, Novus Biological, Littleton, CO, USA), GAPDH (GTX100118, GeneTex, Alton Pkwy, Irvine, CA, USA), and α-tubulin (Sigma-Aldrich).

Techniques: Reverse Transcription, Inhibition, Control, Expressing, Western Blot, Software

Journal: Cancers

Article Title: Short-Term TERT Inhibition Impairs Cellular Proliferation via a Telomere Length-Independent Mechanism and Can Be Exploited as a Potential Anticancer Approach

doi: 10.3390/cancers15102673

Figure Lengend Snippet: p65 inhibition recapitulated the effects of TERT inhibition on MYC and the cell cycle. ( A , B ) Representative Western blots showing total protein levels of p-p65 and MYC in 4134/Late ( A ) and BL41 ( B ) cells upon treatment with Ammonium pyrrolidine dithiocarbamate (PDTC) at the indicated concentrations for 24 h. α-tubulin was used as a loading control. The original Western blots are shown in . Graphs on the right show the values in arbitrary units of densitometric analysis performed with ImageJ software. Data represent the mean and SD (bar) from two separate experiments. ( C , D ) 4134L/Late ( C ) and BL41 ( D ) cells were treated with 30 μM BIBR, PDTC at indicated concentrations, or DMSO as a control for 24 h, labelled with propidium iodide (PI) and analysed by flow cytometry for cell cycle profiles. Panels from a representative experiment are shown. Graphs on the right show percentages of cells in G1, S, and G2/M-phases, respectively. Values show the means and SD (bar) of three separate experiments. A significant difference between values in BIBR-treated or PDTC-treated cells vs. DMSO-treated cells is shown: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The expression of TERT, MYC, p65, phosphorylated p65 (p-p65), cyclin-dependent kinase inhibitor 1A (CDKN1A, P21), telomeric repeat binding factor 2 (TRF2), and α-tubulin were evaluated by the following antibodies: TERT (ab32020, Abcam, Cambridge, UK; abx120550, Abexxa, Arlington, TX, USA), NF-κB p65 (ab32536, Abcam), phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), MYC (ab32072, Abcam), P21 (ab109520, Abcam), TRF2 (NB110-57130, Novus Biological, Littleton, CO, USA), GAPDH (GTX100118, GeneTex, Alton Pkwy, Irvine, CA, USA), and α-tubulin (Sigma-Aldrich).

Techniques: Inhibition, Western Blot, Control, Software, Flow Cytometry

Journal: Cancers

Article Title: Short-Term TERT Inhibition Impairs Cellular Proliferation via a Telomere Length-Independent Mechanism and Can Be Exploited as a Potential Anticancer Approach

doi: 10.3390/cancers15102673

Figure Lengend Snippet: BIBR treatment downregulated the expression of zebrafish myca and mycb and upregulated p21 . Twelve hours post fertilization (hpf), zebrafish wild-type (WT) and tert mutant ( tert hu3430/hu3430 ; tert −/−) embryos were treated with 2 μM BIBR or DMSO as a control for 12 h, from 12 to 24 hpf. Levels of relative mRNA expression for the indicated genes in WT ( A – C ) and tert mutant ( D – F ) embryos are shown. Data represent the mean and SD (bar) from three separate experiments. ( G ) Representative Western blots showing total protein levels of Myc in WT and tert mutant zebrafish embryos upon treatment with 2 μM BIBR for 12 h. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as a loading control. The original Western blots are shown in . The graph shows the values in arbitrary units of densitometric analysis performed with ImageJ software. Data represent the mean and SD (bar) from two separate experiments. A significant difference between values in BIBR-treated embryos vs. DMSO-treated embryos is shown: * p < 0.05; *** p < 0.001; ns: not significant.

Article Snippet: The expression of TERT, MYC, p65, phosphorylated p65 (p-p65), cyclin-dependent kinase inhibitor 1A (CDKN1A, P21), telomeric repeat binding factor 2 (TRF2), and α-tubulin were evaluated by the following antibodies: TERT (ab32020, Abcam, Cambridge, UK; abx120550, Abexxa, Arlington, TX, USA), NF-κB p65 (ab32536, Abcam), phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), MYC (ab32072, Abcam), P21 (ab109520, Abcam), TRF2 (NB110-57130, Novus Biological, Littleton, CO, USA), GAPDH (GTX100118, GeneTex, Alton Pkwy, Irvine, CA, USA), and α-tubulin (Sigma-Aldrich).

Techniques: Expressing, Mutagenesis, Control, Western Blot, Software

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: A QPCR results showed that CB1 was downregulated in colorectal cancer cells. B QPCR results showed that EGFR was upregulated in colorectal cancer cells. C, D Western blot analysis showed that CB1 was downregulated and EGFR was upregulated in colorectal cancer cells. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Western Blot

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: A Western blot assay tested the protein level of CB1 after ACEA and AM251 treatment. B Western blot analysis showed that CB1 activation significantly decreased the expression of EGFR while its inhibition increased the expression of EGFR. C, D Colony forming assay showed that CB1 activation suppressed colony formation of colorectal cancer cells, while its inhibition promoted colony formation. E, F Wound healing test showed that CB1 activation prevented cell migration while CB1 inhibition enhanced cell migration of colorectal cancer cells. G, H Transwell invasion assay demonstrated that CB1 activation blocked cell invasion while CB1 inhibition enhanced cell invasion of colorectal cancer cells. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Western Blot, Activation Assay, Expressing, Inhibition, Migration, Transwell Invasion Assay

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: Cells were transfected with EGFR plasmids and simultaneously treated with ACEA. A QPCR analysis showed that EGFR plasmids transfection increased the expression of EGFR in SW480 and SW620 cells. B , C Colony-forming assay demonstrated that EGFR overexpression blocked the proliferation suppression induced by CB1 activation. D , E Wound healing test showed that EGFR overexpression counteracted the migration inhibition induced by CB1 activation. F , G Transwell invasion assay showed that EGFR overexpression prevented the decrease in cell invasion caused by CB1 activation. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Transfection, Expressing, Over Expression, Activation Assay, Migration, Inhibition, Transwell Invasion Assay

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: Colorectal cancer cells were transfected with EGFR plasmids and simultaneously treated with ACEA. Then PMA induced THP-1 cells were co-cultured with the culture medium of colorectal cancer cells. A – F QPCR results demonstrated that EGFR overexpression blocked CB1 activation caused increase in the expression of IL-6 and TNF-α, and decrease in IL-10, CCL22, Arg-1 and CD206. G – J ELISA assay showed that EGFR overexpression blocked CB1 activation caused increase in the release of IL-6 and TNF-α, and decrease in the release of IL-10 and CCL22. K , L Western blot showed that EGFR overexpression prevented the decrease in the expression of Arg-1 and CD206. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Transfection, Cell Culture, Over Expression, Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: Colorectal cells were transfected with plasmids bearing shRNA against EGFR and simultaneously treated with AM251. Culture medium for these colorectal cells were taken to incubate PMA-induced THP-1 cells. A QPCR analysis showed that sh-EGFR effectively suppressed the expression of EGFR. B , C Colony formation test demonstrated that EGFR knockdown reversed the enhancement in colony formation induced by AM251. D – I QPCR results demonstrated that EGFR knockdown reversed the CB1 inhibition caused decrease in the expression of IL-6 and TNF-α, and increase in IL-10, CCL22, Arg-1 and CD206. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Transfection, shRNA, Expressing, Knockdown, Inhibition

Journal: Cell Death Discovery

Article Title: Cannabinoid Receptor-1 suppresses M2 macrophage polarization in colorectal cancer by downregulating EGFR

doi: 10.1038/s41420-022-01064-8

Figure Lengend Snippet: SW480 cells were subcutaneously injected into nude mice and the tumors were treated with control or ACEA (1.5 mg/kg/d). A CB1 activation suppressed tumor growth. Representative tumors ( B ) and tumor weight ( C ) from different experimental groups. D QPCR analysis showed that CB1 activation increased the expression of IL-6 and TNF-α, and decreased the expression of IL-10, CCL-22, Arg-1 and CD206. E Western blot analysis showed that the expression of EGFR, Arg-1 and CD206 were decreased in the tumors of ACEA-treated mice. * P < 0.05; ** P < 0.01.

Article Snippet: After blocking with 5% BSA, membranes were incubated with primary antibodies: Arg-1, 93668, CST (Boston, Massachusetts, USA); CD206, ab64693, Abcam (Cambridge, MA, USA); CB1, ab259323 Abcam; CB2, ab3561 Abcam; EGFR, ab52894, Abcam; β-actin, ab8226, Abcam at 4 °C overnight.

Techniques: Injection, Control, Activation Assay, Expressing, Western Blot

Journal: The FASEB Journal

Article Title: G9a promotes cell proliferation and suppresses autophagy in gastric cancer by directly activating mTOR

doi: 10.1096/fj.201900233rr

Figure Lengend Snippet: Figure 2. Down-regulation of G9a represses cell proliferation of GC. A) Western blotting and real-time qPCR assays were performed to detect the G9a expression in 3 G9a-knockdown GC cell lines. Tubulin was used as a loading control. B) G9a knockdown repressed the growth and proliferation of HGC-27, MKN-45, and SGC-7901 cells. Cell viability was detected using CCK8 assays. C) GC cells were treated with BIX01294 and analyzed for cell viability using CCK8 assays. D) Ki67 immunofluorescence assays were performed after G9a knockdown or inhibition. Cells were treated with 7 mM BIX01294 for 48 h. Representative images show immunofluorescence. Scale bars, 20 mm. All data are shown as means 6 SD; n = 3. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: G9a (ab40542), Ki67 (ab92742), Unc-51–like autophagyactivating kinase 1 (ULK1; ab128859), Beclin-1 (ab207612), p62/ SQSTM1 (ab207305), mTOR (ab32028), H3K9me1 (ab9045), H3K9me2 (ab1220), and p-H3S10 (5176) antibodies were purchased from Abcam (Cambridge, MA, USA). p(Ser2448)-mTOR (D9C2), CyclinA2 (BF683), CyclinB1 (D5C10), p(Ser757)-ULK1 (D7O6U), p70S6K (D5U1O), CDK1 (POH1), and LC3B (D11) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Knockdown, Control, Inhibition

Journal: The FASEB Journal

Article Title: G9a promotes cell proliferation and suppresses autophagy in gastric cancer by directly activating mTOR

doi: 10.1096/fj.201900233rr

Figure Lengend Snippet: Figure 3. Down-regulation of G9a represses colony formation in vitro and tumor formation of GC cells in vivo. A, B) The effects of G9a on the colony formation in 3 G9a-knockdown GC cell lines. Scale bars, 20 mm. The colony numbers in plate were quantified. C, D) The tumor growth curve and tumor weight of G9a knockdown GC cells injected into NOD-SCID mice. E) Immunohistochemical (IHC) staining of G9a expression (left) and Ki67 expression (right). Scale bars, 20 mm. F) The quantification of G9a-positive cells (left) and Ki67-positive cells (right). All data are shown as means 6 SD; n = 3. **P , 0.01, ***P , 0.001.

Article Snippet: G9a (ab40542), Ki67 (ab92742), Unc-51–like autophagyactivating kinase 1 (ULK1; ab128859), Beclin-1 (ab207612), p62/ SQSTM1 (ab207305), mTOR (ab32028), H3K9me1 (ab9045), H3K9me2 (ab1220), and p-H3S10 (5176) antibodies were purchased from Abcam (Cambridge, MA, USA). p(Ser2448)-mTOR (D9C2), CyclinA2 (BF683), CyclinB1 (D5C10), p(Ser757)-ULK1 (D7O6U), p70S6K (D5U1O), CDK1 (POH1), and LC3B (D11) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vitro, In Vivo, Knockdown, Injection, Immunohistochemical staining, Immunohistochemistry, Expressing