Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 1. TMCO1 presents high expression in ovarian carcinoma and is linked with undesirable clinical outcomes. (A) Box plot shows the RNA expression of TMCO1 in 426 ovarian carcinoma tissues (T) and 88 normal tissues (N) in the integrated TCGA and GTEx data. *P<0.05. (B, C) Kaplan-Meier analysis depicts the survival diffe rence in ovarian carcinoma patients possessing high TMCO1 expres sion relative to those possessing low TMCO1 expression in the (B) GSE26712 and (C) GSE27651 cohorts. (D) Immunohistochemistry shows the expression and distribution of TMCO1 in adjacent normal tissues and ovarian carcinoma tissues. Scale bar, 20 μm. (E) A bar graph visualizes TMCO1 expression in two groups following immu nohistochemistry results. **P<0.01. (F) Immunofluorescence exa mines the expression and distribution of TMCO1 in adjacent normal tissues and ovarian carcinoma tissues. Scale bar, 20 μm. (G) A bar graph shows TMCO1 expression in two groups in accordance with immunofluorescence results. ***P<0.001.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Expressing, RNA Expression, Immunohistochemistry, Immunofluorescence

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 2. TMCO1 up-regulation enhances proliferation of cisplatin- sensitive as well as resistant ovarian carcinoma cells. (A, B) Western blot verifies TMCO1 expression in SK-OV-3 cells administrated with specific shRNAs against TMCO1. (C, D) Western blot validates the overexpression of TMCO1 in SK-OV-3 cells. (E, F) The clonogenic assay shows the colonies of SK-OV-3 cell line with TMCO1-knoc kout or overexpressed plasmids. (G, H) Clonogenic assay displays the colonies of SK-OV-3-CDDP cell line with TMCO1-knockout or overexpressed plasmids. **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Western Blot, Expressing, Over Expression, Clonogenic Assay, Knock-Out

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 3. High TMCO1 expression facilitates migration of cisplatin-sen sitive as well as resistant ovarian carcinoma cells. (A, B) A wound- healing experiment was applied to examine the migration capacity of SK-OV-3 cells subjected to TMCO1-knockout or overexpressed plasmids. Scale bar, 200 μm. (C, D) A wound wound-healing expe riment was applied to evaluate the migrative capacity of SK-OV- 3-CDDP cells subjected to TMCO1-knockout or overexpressed plas mids. Scale bar, 200 μm. (E, F) A Transwell assay was conducted to investigate the number of migrative cells of SK-OV-3 cells subjected to TMCO1-knockout or overexpressed plasmids. Scale bar, 50 μm. (G, H) Transwell assay was presented for assessing the number of migrative SK-OV-3-CDDP cells with TMCO1-knockout or overex pressed plasmids. Scale bar, 50 μm. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Expressing, Migration, Knock-Out, Transwell Assay

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 4. TMCO1 regulated calcium levels and cytoskeletal remodeling in SK-OV-3 and SK-OV-3-CDDP cells. (A,B) Representative photo graphs of cytosolic Ca2+ level in SK-OV-3 cells and SK-OV-3-CDDP via Fluo-4 AM staining were displayed. Bar, 20 μm. (C,D) Phalloidin staining (Green) after TMCO1-knockout or overexpressed plasmids. Bar, 20 μm.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Staining, Knock-Out

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 5. The interaction of TMCO1 with CALR and VDAC1 and its influence on the expression of EMT markers. (A-H) Western blotting was conducted for examining (B) TMCO1, (C) VDAC1, (D) CALR, (E) Vimentin, (F) β-catenin, (G) N-cadherin, and (H) E-cadherin ex pression in SK-OV-3 cells subjected to TMCO1-knockout or overex pressed plasmids. (I-P) Western blotting was conducted to examine (J) TMCO1, (K) VDAC1, (L) CALR, (M) Vimentin, (N) β-catenin, (O) N-cadherin, and (P) E-cadherin expression in SK-OV-3-CDDP cell line subjected to TMCO1-knockout or overexpressed plasmids. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Expressing, Western Blot, Knock-Out

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 6. The interaction of TMCO1 with CALR and VDAC1 and its influence on the expression of EMT markers. (A, B) Representative images of immunofluorescence for SK-OV-3 or SK-OV-3-CDDP cell lines. Bar scale, 20 μm. (C-G) The expression levels of (C) TMCO1, (D) CALR, (E) VDAC1, (F) N-cadherin, and (G) E-cadherin were quantified in SK-OV-3 cells subjected to TMCO1-knockout or ove rexpressed plasmids. (H-L) The expression levels of (H) TMCO1, (I) CALR, (J) VDAC1, (K) N-cadherin, or (L) E-cadherin were quanti fied for SK-OV-3-CDDP cell line with TMCO1-knockout or overex pressed plasmids. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Expressing, Immunofluorescence, Knock-Out

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 7. The interaction of TMCO1 with CALR and VDAC1. (A-L) Western blotting was conducted to examine (B) TMCO1, (C)CALR and( D) VDAC1 expression with CALR-siRNA and VDAC1-siRNA transfections. (E-H) Western blotting was conducted for examining (F) TMCO1, (G) CALR, (H) VDAC1 in SK-OV-3 cells. (I-L) Wes tern blotting was conducted for examining (J) TMCO1, (K) CALR, (L) VDAC1 With TMCO1 overexpression or CALR-siRNA and VDAC1-siRNA transfections in SK-OV-3-CDDP cells.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Western Blot, Expressing, Transfection, Over Expression

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 8. The interaction of TMCO1 with CALR and VDAC1. (A-D) Western blotting was conducted for examining (B) TMCO1, (C) CALR and (D) VDAC1 expression with CALR-siRNA +VDAC1- siRNA or sh-TMCO1 with CALR-siRNA and VDAC1-siRN in SK- OV-3 cells. (E-H) Western blotting was conducted for examining (F) TMCO1, (G) CALR and (H)VDAC1 expression with CALR-siRNA +VDAC1-siRNA or sh-TMCO1 with CALR-siRNA and VDAC1- siRNA in SK-OV-3-CDDP cells.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Western Blot, Expressing

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 9. Targeting TMCO1 inhibits the growth of SK-OV-3 and SK- OV-3-CDDP cells in xenograft models. (A) Photographs of nude mouse models with sh-NC-transfected SK-OV-3 cell line (control group), mice injected with sh-NC-transfected SK-OV-3-CDDP cell line (CDDP group), mice injected with sh-TMCO1-transfected SK- OV-3 cells (sh-TMCO1 group), mice injected with sh-TMCO1-trans fected SK-OV-3-CDDP cells (CDDP + sh-TMCO1 group). (B) Photo graphs of tumors from above four groups. (C) Tumor growth curves in each group. Compared with control group, *p<0.05; ****P<0.0001. Compared with CDDP group, ####P<0.0001. (D, E) Immunohisto chemistry examining the expression of CD34 in tumors from four groups. Bar scale, 20 μm. (F, G) Immunofluorescence detecting CD34 expression in tumors from four groups. Bar scale, 20 μm. *P<0.05; ***P<0.001; ****P<0.0001.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Transfection, Control, Injection, Immunohistochemistry, Expressing, Immunofluorescence

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 10. Targeting TMCO1 inhibited the expression of CALR, VDAC1 and EMT markers in xenograft tumors. (A-K) Western blot was ap plied for examining (B) TMCO1, (C) VDAC1, (D) CALR, (E) Ki-67, (F) MMP2, (G) MMP9, (H) N-cadherin, (I) Vimentin, (J) β-catenin, or (K) E-cadherin expression within xenograft tumors developed by sh-NC or sh-TMCO1-transfected SK-OV-3 or SK-OV-3-CDDP cell line. **P<0.05; ***P<0.001; ****P<0.0001.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Expressing, Western Blot, Transfection

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

Article Title: TMCO1 regulates cell proliferation, metastasis and EMT signaling through CALR, promoting ovarian cancer progression and cisplatin resistance.

doi: 10.14715/cmb/2024.70.1.14

Figure Lengend Snippet: Fig. 11. The role of TMCO1, CALR, and VDAC1 in xenograft tu mors. (A, B) Representative images of (A) immunohistochemistry as well as (B) immunofluorescence in xenograft tumors. Bar scale, 20 μm. (C-I) Expression of (C) TMCO1, (D) VDAC1, (E) CALR, (F) β-catenin, (G) Vimentin, (H) N-cadherin, or (I) E-cadherin was quantified in xenograft tumors developed by sh-NC or sh-TMCO1- transfected SK-OV-3 or SK-OV-3-CDDP cell line according to immu nohistochemistry. (J-P) Expression of (J) TMCO1, (K) VDAC1, (L) CALR, (M) β-catenin, (N) Vimentin, (O) N-cadherin, or (P) E-cadhe rin was quantified within xenograft tumors developed by sh-NC or sh- TMCO1-transfected SK-OV-3 or SK-OV-3-CDDP cell line according to immunofluorescence. **P<0.05; ***P<0.001; ****P<0.0001.

Article Snippet: The membranes were sealed in 5% skim milk lasting one hour at room temperature, as well as incubated by primary antibody against TMCO1 (1:500; 27757-1-AP; Proteintech, Rosemont, IL, USA), β-catenin (1:2000; 51067-2-AP; Proteintech, Rosemont, IL, USA), VDAC1 (1:1000; 55259-1-AP; Proteintech, Rosemont, IL, USA), CALR (1:1000; 27298-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (1:1000; 66366- 1-Ig; Proteintech, Rosemont, IL, USA), MMP9 (1:1000; 10375-2-AP; Proteintech, Rosemont, IL, USA), N-cadherin (1:1000; 22018-1-AP; Proteintech, Rosemont, IL, USA), E-cadherin (1:1000; 20874-1-AP; Proteintech, Rosemont, IL, USA), Vimentin (1:2000; 10366-1-AP; Proteintech, Rosemont, IL, USA), Ki-67 (1:1000; ab16667; Abcam, Cambridge, MA, USA) or GAPDH (1:5000; 10494-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C.

Techniques: Immunohistochemistry, Immunofluorescence, Expressing, Transfection

Journal: BMC cancer

Article Title: Integrative machine learning frameworks to uncover specific protein signature in neuroendocrine cervical carcinoma.

doi: 10.1186/s12885-025-13454-z

Figure Lengend Snippet: Fig. 4 IHC staining of kNsDEPs in normal cervix specimens, paracancerous and tumor tissues of CSCC and NECC. (A-C) Representative IHC images of SCGN (A), CACYBP (B), and CAP2 (C) in the indicated specimens (n = 3). (D-F) Quantitative statistical analysis of protein levels of SCGN (D), CACYBP (E), and CAP2 (F) according to (A-C) and Figure S5. (n = 3) (*p < 0.05, **p < 0.01, ***p < 0.001; ns: not significant)

Article Snippet: The rabbit polyclonal antibody targeting CACYBP was obtained from Proteintech, USA (11745- 1-AP), with a primary antibody dilution of 1:200 for CACYBP.

Techniques: Immunohistochemistry

Journal: BMC cancer

Article Title: Integrative machine learning frameworks to uncover specific protein signature in neuroendocrine cervical carcinoma.

doi: 10.1186/s12885-025-13454-z

Figure Lengend Snippet: Fig. 5 Nomogram models based on kNsDEPs. (A) Nomogram model for NECC prediction based on SCGN, CAP2, and CACYBP. (B) The AUC score of (A). (C) Nomogram model for NECC prediction from TCGA based on SCGN, CAP2, and CACYBP. (D) The AUC score of (C)

Article Snippet: The rabbit polyclonal antibody targeting CACYBP was obtained from Proteintech, USA (11745- 1-AP), with a primary antibody dilution of 1:200 for CACYBP.

Techniques:

Journal: Cellular microbiology

Article Title: Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

doi: 10.1111/cmi.13033

Figure Lengend Snippet: Identification of Cholix-interaction proteins on HepG2 cells and immortalised human hepatocytes. (a) After biotinylation of surface proteins, hepatocytes were solubilised with RIPA buffer and immunoprecipitated with heat-inactivated or catalytically inactive mutant Cholix (E581A; C) as described in Section 4. Cholix-binding proteins were detected using streptavidin-HRP. (b) After trypsin hydrolysis, LC-MS/MS analysis of 30-kDa and 33-kDa proteins was performed. The protein had sequences identical to that of proteins, PHB1 and PHB2. (c) Proteins from HepG2 or hepatocytes were immunoprecipitated with Cholix as described above, separated by SDS-PAGE and transferred to PVDF membranes, which were reacted with anti-PHB1 (left) or anti-PHB2 antibodies (right). TCL; total cell lysate. (d) Purified wild-type Cholix (0.5 μg) was incubated for 4 hr with GST-, GST-fused PHB1, or GST-fused PHB2 conjugated beads and then washed with PBS three times. After SDS-PAGE, proteins were transferred to PVDF membranes and visualised by Western blotting using GST-HRP or anti-Cholix antibodies

Article Snippet: For Western blot analysis, anti-cleaved PARP (9542), anti-cleaved Caspase 8 (9496), anti-cleaved Caspase 3 (9664), anti-Bax (2772), and anti-Bak (3814), antibodies were purchased from Cell Signaling Technology; anti-Bax (610982) antibodies were from BD Bioscience; anti-PHB1 (10787-1-AP) and anti-PHB2 (12295-1-AP) antibodies were from ProteinTech; anti-PHB1 (sc-377037), anti-PHB2 (sc-133094), anti-TOM20 (F-10) (sc-17764) monoclonal antibodies and anti-eEF2 (H-118; sc-118), anti-Drp1 (H-300; sc-32898) antibodies were from Santa Cruz Biotechnology; anti-Bak (Ab-2; TC-102) monoclonal antibody was from CALBIOCHEM; anti-ROCK1 (GTX113266) and anti-GAPDH (GTX100118) antibodies were from GeneTex; anti-FLAG antibody (018-22381) was from WAKO Pure Chemical; anti-Myc tag antibody (M192-3) was from MBL; HRP-conjugated anti-mouse IgG (HAF007), anti-rabbit IgG (HAF008) antibodies, and Z-VAD-FMK were purchased from R&D Systems; anti-GST HRP (RPN1236) and Streptavidin-HRP were obtained from GE Healthcare Life Sciences.

Techniques: Immunoprecipitation, Mutagenesis, Binding Assay, Liquid Chromatography with Mass Spectroscopy, SDS Page, Purification, Incubation, Western Blot

Journal: Cellular microbiology

Article Title: Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

doi: 10.1111/cmi.13033

Figure Lengend Snippet: Effect of Cholix on prohibitin (PHB) expression and localisation in hepatocytes. (a) Hepatocytes were treated for the indicated time points with 100 μg/ml cycloheximide (CHX), 5 μg/ml PEA, mutant Cholix(E581A; mt) or wild-type (wt) Cholix. Then, cells were lysed with 1xSDS sample buffer for immunoblotting with the indicated antibodies. GAPDH was used as an internal control. Quantification of PHB1 and PHB2 levels in hepatocytes was performed by densitometry (bottom panel). GAPDH was used as a loading control. Data are presented as mean ± SD of values from three experiments and significance is *p < .05. Experiments were repeated three times with similar results. (b) Hepatocytes were incubated for 12 hr with mt or wt Cholix (5 μg/ml), and total RNA was extracted as described in Section 4. PHB1 and PHB2 mRNA were measured by real-time qPCR. Data are shown as mean ± SD of values from two experiments. (c) Hepatocytes (5 × 104 cells/well) in 12-well plates were treated with wt Cholix (5 μg/ml) for the indicated times. Then, cells were fixed and visualised using anti-PHB1 or PHB2 antibodies; mitochondria were seen with MitoTracker as described in Section 4. Nuclei were stained with DAPI. Experiments were repeated three times with similar results. (d) Hepatocytes were treated for 12 hr with 5 μg/ml mt or wt Cholix. Then, cells were lysed with RIPA buffer and then immunoprecipitated with control rabbit IgG (C) or anti-PHB1 monoclonal antibody (P1). The complexes were lysed with 1xSDS sample buffer and analysed by immunoblotting with the indicated antibodies. TCL: total cell lysate. Experiments were repeated three times with similar results. (e) Hepatocytes (5 × 104 cells/well) in 12-well plates were incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were fixed, and PHB1 and PHB2 were visualised as described in Section 4. Nuclei were stained with DAPI. Experiments were repeated three times with similar results

Article Snippet: For Western blot analysis, anti-cleaved PARP (9542), anti-cleaved Caspase 8 (9496), anti-cleaved Caspase 3 (9664), anti-Bax (2772), and anti-Bak (3814), antibodies were purchased from Cell Signaling Technology; anti-Bax (610982) antibodies were from BD Bioscience; anti-PHB1 (10787-1-AP) and anti-PHB2 (12295-1-AP) antibodies were from ProteinTech; anti-PHB1 (sc-377037), anti-PHB2 (sc-133094), anti-TOM20 (F-10) (sc-17764) monoclonal antibodies and anti-eEF2 (H-118; sc-118), anti-Drp1 (H-300; sc-32898) antibodies were from Santa Cruz Biotechnology; anti-Bak (Ab-2; TC-102) monoclonal antibody was from CALBIOCHEM; anti-ROCK1 (GTX113266) and anti-GAPDH (GTX100118) antibodies were from GeneTex; anti-FLAG antibody (018-22381) was from WAKO Pure Chemical; anti-Myc tag antibody (M192-3) was from MBL; HRP-conjugated anti-mouse IgG (HAF007), anti-rabbit IgG (HAF008) antibodies, and Z-VAD-FMK were purchased from R&D Systems; anti-GST HRP (RPN1236) and Streptavidin-HRP were obtained from GE Healthcare Life Sciences.

Techniques: Expressing, Mutagenesis, Western Blot, Control, Incubation, Staining, Immunoprecipitation

Journal: Cellular microbiology

Article Title: Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

doi: 10.1111/cmi.13033

Figure Lengend Snippet: Cholix-induced apoptosis is enhanced in PHB-knockdown hepatocytes. (a) Control (NC) and PHB1 or PHB2 siRNA-transfected hepatocytes were labeled with biotin, lysed with RIPA buffer, and then immunoprecipitated with Cholix or avidin-beads. The complexes were lysed with 1xSDS sample buffer and analysed by immunoblotting with the indicated antibodies. (b) The indicated siRNA-transfected hepatocytes were incubated for 8 hr with mt or wt Cholix (5 μg/ml), lysed with 1xSDS sample buffer and analysed by immunoblotting with antibodies. Quantification of cPARP, PHB1, or PHB2 levels in hepatocytes was performed by densitometry (right panel). GAPDH was used as a loading control. Data are presented as mean ± SD of values from three experiments, and significance is *p < .05. Experiments were repeated three times with similar results. (c) The indicated siRNA-transfected hepatocytes were incubated for 8 hr with CHX (100 μg/ml) or PEA (5 μg/ml), lysed with 1xSDS sample buffer, and analysed by immunoblotting with antibodies. Quantification of cPARP level in hepatocytes was performed by densitometry (bottom panel). GAPDH was used as a loading control. Data are presented as mean ± SD of values from three experiments. n.s.: not significant. (d) The indicated siRNA-transfected Hepatocytes (5 × 104 cells/well) in 12-well plates were incubated for 24 hr with wt or mt Cholix (5 μg/ml). Cells were fixed with 4% PFA and then visualised with anti-actin (green) or α-tubulin (red) antibodies. Nuclei were stained with DAPI. The bottom panel of each group represents enlarged version of the white square in middle panel. Experiments were repeated two times with similar results

Article Snippet: For Western blot analysis, anti-cleaved PARP (9542), anti-cleaved Caspase 8 (9496), anti-cleaved Caspase 3 (9664), anti-Bax (2772), and anti-Bak (3814), antibodies were purchased from Cell Signaling Technology; anti-Bax (610982) antibodies were from BD Bioscience; anti-PHB1 (10787-1-AP) and anti-PHB2 (12295-1-AP) antibodies were from ProteinTech; anti-PHB1 (sc-377037), anti-PHB2 (sc-133094), anti-TOM20 (F-10) (sc-17764) monoclonal antibodies and anti-eEF2 (H-118; sc-118), anti-Drp1 (H-300; sc-32898) antibodies were from Santa Cruz Biotechnology; anti-Bak (Ab-2; TC-102) monoclonal antibody was from CALBIOCHEM; anti-ROCK1 (GTX113266) and anti-GAPDH (GTX100118) antibodies were from GeneTex; anti-FLAG antibody (018-22381) was from WAKO Pure Chemical; anti-Myc tag antibody (M192-3) was from MBL; HRP-conjugated anti-mouse IgG (HAF007), anti-rabbit IgG (HAF008) antibodies, and Z-VAD-FMK were purchased from R&D Systems; anti-GST HRP (RPN1236) and Streptavidin-HRP were obtained from GE Healthcare Life Sciences.

Techniques: Knockdown, Control, Transfection, Labeling, Immunoprecipitation, Avidin-Biotin Assay, Western Blot, Incubation, Staining

Journal: Cellular microbiology

Article Title: Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes

doi: 10.1111/cmi.13033

Figure Lengend Snippet: ROCK1 participates in a Cholix-induced apoptotic pathway. (a) Hepatocytes were pretreated for 30 min with or without 0, 30, 60 μM Y27632 and then incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were lysed with 1xSDS sample buffer for immunoblotting with anti-cleaved PARP (cPARP) or GAPDH antibodies. A blot representative of three independent experiments is shown. (b) Hepatocytes were pretreated for 30 min with or without 60 μM Y27632 and then incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were lysed with 1xSDS sample buffer for immunoblotting with anti-cPARP, ROCK1, PHB1, PHB2, or GAPDH antibodies. A blot representative of three separate experiments is shown. (c) The indicated siRNA-transfected hepatocytes were incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were lysed with 1xSDS sample buffer for immunoblotting with the indicated antibodies. A blot representative of three independent experiments is shown. (d) Hepatocytes were pretreated for 30 min with or without 60μM Y27632 and then incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were treated with CellROX Deep Red reagent before fixation with 4% PFA and staining with anti-TOM20 (green) or anti-Drp1 (Red) antibodies. Nuclei were stained with DAPI as described in Section 4. Experiments were repeated three times with similar results. (e) The indicated siRNA-transfected hepatocytes were incubated for 12 hr with mt or wt Cholix (5 μg/ml). Then, cells were lysed with 1xSDS sample buffer for immunoblotting with the indicated antibodies. A blot representative of three independent experiments is shown. (f) The indicated siRNA-transfected hepatocytes were incubated for 12 hr with mt or wt Cholix (5 μg/ml) in the presence or absence of 20 μM Z-VAD-FMK. Then, cells were lysed with 1xSDS sample buffer for immunoblotting with the indicated antibodies. A blot representative of three independent experiments is shown.

Article Snippet: For Western blot analysis, anti-cleaved PARP (9542), anti-cleaved Caspase 8 (9496), anti-cleaved Caspase 3 (9664), anti-Bax (2772), and anti-Bak (3814), antibodies were purchased from Cell Signaling Technology; anti-Bax (610982) antibodies were from BD Bioscience; anti-PHB1 (10787-1-AP) and anti-PHB2 (12295-1-AP) antibodies were from ProteinTech; anti-PHB1 (sc-377037), anti-PHB2 (sc-133094), anti-TOM20 (F-10) (sc-17764) monoclonal antibodies and anti-eEF2 (H-118; sc-118), anti-Drp1 (H-300; sc-32898) antibodies were from Santa Cruz Biotechnology; anti-Bak (Ab-2; TC-102) monoclonal antibody was from CALBIOCHEM; anti-ROCK1 (GTX113266) and anti-GAPDH (GTX100118) antibodies were from GeneTex; anti-FLAG antibody (018-22381) was from WAKO Pure Chemical; anti-Myc tag antibody (M192-3) was from MBL; HRP-conjugated anti-mouse IgG (HAF007), anti-rabbit IgG (HAF008) antibodies, and Z-VAD-FMK were purchased from R&D Systems; anti-GST HRP (RPN1236) and Streptavidin-HRP were obtained from GE Healthcare Life Sciences.

Techniques: Incubation, Western Blot, Transfection, Staining

Journal: Nature Communications

Article Title: In-situ cross-linking mass spectrometry reveals compartment-specific proteasomal interactions and structural heterogeneity

doi: 10.1038/s41467-025-65752-6

Figure Lengend Snippet: A Complex model of Rpt5 ( P17980 , residues 166–439) and PSMD9 ( O00233 ), with full torsional freedom allowed for the linker residues (115–134) between PSMD9’s two domains. B AlphaFold model of free PSMD9, with the backbone colored according to pLDDT values (yellow, <70; orange, <50). The arrow indicates the direction of domain closure upon binding to Rpt5. C AlphaFold-modeled complex structure between Rpn5 ( O00232 ), EIF3M ( Q7L2H7 ), and Rpn8 ( P51665 ), shown in two different perspectives. The sequence and structure comparison between EIF3M and Rpn9 ( Q9UNM6 ) are shown in Supplementary Fig. . Dashed lines indicate experimental intermolecular cross-links, with associated Lys residues denoted.

Article Snippet: The following antibodies were used: EIF3M (Proteintech, 11423-1-AP), RPS5 (Santa Cruz, sc-390935), and a control IgG antibody (Proteintech, 30000-0-AP, 1:200 dilution).

Techniques: Binding Assay, Sequencing, Comparison

Journal: Nature Communications

Article Title: In-situ cross-linking mass spectrometry reveals compartment-specific proteasomal interactions and structural heterogeneity

doi: 10.1038/s41467-025-65752-6

Figure Lengend Snippet: A Co-immunoprecipitation confirms that EIF3M interacts with Rpn11, a proteasomal subunit, and with Rps5, a component of the 48S translation initiation complex. Three biological repeats were performed, with the representative micrograph shown here (raw micrographs provided in the Source data). B Immunofluorescence microscopy reveals co-localization of EIF3M with both Rps5 and Rpn11in the cytoplasm and nucleus. Fluorescence intensity measurements indicate that EIF3M/Rpn11 levels are approximately 3–5 times higher in the cytoplasm compared to the nucleus.

Article Snippet: The following antibodies were used: EIF3M (Proteintech, 11423-1-AP), RPS5 (Santa Cruz, sc-390935), and a control IgG antibody (Proteintech, 30000-0-AP, 1:200 dilution).

Techniques: Immunoprecipitation, Immunofluorescence, Microscopy, Fluorescence