Journal: Translational Lung Cancer Research

Article Title: Senescence marker protein 30 inhibits tumor growth by reducing HDAC4 expression in non-small cell lung cancer

doi: 10.21037/tlcr-21-982

Figure Lengend Snippet: Effect of SMP30 overexpression on proliferative ability of A549 and H1299 cells, and tumor growth in H1299 cell tumor xenografts. (A) Relative protein levels of SMP30 in cell lines as determined by Western blot. β-actin was used as a loading control. (B) Representative Western blot results of Flag, SMP30, c-Myc and CyclinD1 in LV-SMP30 A549 and LV-SMP30 H1299 cells. (C) Growth curves of the A549 and H1299 cells. Cell viability was detected by CCK-8 assay and expressed as optical density values. (D) Representative images and results of the colony formation assay. Colonies were visualized by crystal violet staining. (E) Representative images (×20) and statistical analysis of EdU incorporation assay. The results are presented as the ratio between the number of EdU-positive cells (red fluorescence) and the total number of Hoechst 33342-stained cells (blue fluorescence). (F) Representative results of tumor weight changes after subcutaneous injection of H1299 cells. Photographs show tumor xenograft morphologies in the LV-control and LV-SMP30 H1299 groups. (G) Representative Western blot results of Flag, SMP30, c-Myc and CyclinD1 in H1299 xenograft tumor tissues. β-actin was used as a Western blot loading control. The values below the Western blot band represent the relative gray values. All the data are shown as the mean ± SD. Student’s t-test, *, P<0.05 vs. LV-control group. LV, lentivirus; SMP30, senescence marker protein 30.

Article Snippet: Antibodies against SMP30 (1:1,000, 17947-1-AP, Proteintech), against HDAC4 (1:1,000, Proteintech Cat# 17449-1-AP, RRID: AB_2118864), anti- CyclinD1 (1:1,000, 60186-1-Ap, Proteintech), anti- c-Myc (1:1,000, #5605, CST) anti- β - actin (1:5,000, ab6276, Abcam), and anti- tubulin (1:1,000, #2148, CST) were used.

Techniques: Over Expression, Western Blot, Control, CCK-8 Assay, Colony Assay, Staining, Fluorescence, Injection, Marker

Journal: Translational Lung Cancer Research

Article Title: Senescence marker protein 30 inhibits tumor growth by reducing HDAC4 expression in non-small cell lung cancer

doi: 10.21037/tlcr-21-982

Figure Lengend Snippet: Effect of HDAC4 level of SMP30 overexpression both in vivo and in vitro, and effect of HDAC4 overexpression on NSCLC cell proliferation. (A) Representative Western blot results of HDAC4, HDAC5, HDAC7 and HDAC9 in LV-SMP30 A549 and LV-SMP30 H1299 cells. (B) Representative Western blot results of HDAC4, HDAC5, HDAC7 and HDAC9 in H1299 xenograft tumor tissues. (C) Relative protein levels of HDAC4 in cell lines as determined by Western blot. (D) Representative Western blot results of Flag, HDAC4, c-Myc and CyclinD1 in LV-HDAC4 A549 and LV-HDAC4 H1299 cells. (E) Representative images and results of colony formation assay. Colonies were visualized by crystal violet staining. (F) Representative images (×20) and statistical analysis of EdU incorporation assay. The results are presented as the ratio between the number of EdU-positive cells (red fluorescence) and the total number of Hoechst 33342-stained cells (blue fluorescence). β-actin was used as a Western blot loading control. The values below the Western blot band represent the relative gray values. All the data are shown as the mean ± SD. Student’s t-test, *, P<0.05 vs. LV-control group. HDAC histone deacetylase; LV, lentivirus; SMP30, senescence marker protein 30; SD, standard deviation; NSCLC, non-small cell lung cancer.

Article Snippet: Antibodies against SMP30 (1:1,000, 17947-1-AP, Proteintech), against HDAC4 (1:1,000, Proteintech Cat# 17449-1-AP, RRID: AB_2118864), anti- CyclinD1 (1:1,000, 60186-1-Ap, Proteintech), anti- c-Myc (1:1,000, #5605, CST) anti- β - actin (1:5,000, ab6276, Abcam), and anti- tubulin (1:1,000, #2148, CST) were used.

Techniques: Over Expression, In Vivo, In Vitro, Western Blot, Colony Assay, Staining, Fluorescence, Control, Histone Deacetylase Assay, Marker, Standard Deviation

Journal: Translational Lung Cancer Research

Article Title: Senescence marker protein 30 inhibits tumor growth by reducing HDAC4 expression in non-small cell lung cancer

doi: 10.21037/tlcr-21-982

Figure Lengend Snippet: Essential requirement for HDAC4 in SMP30-mediated NSCLC cell proliferation. (A) Representative Western blot results of Flag, SMP30, HDAC4 and c-Myc and CyclinD1. (B) Representative images and results of colony formation assay. Colonies were visualized by crystal violet staining. (C) Representative images (×20) and statistical analysis of EdU incorporation assay. The results are presented as the ratio between the number of EdU-positive cells (red fluorescence) and the total number of Hoechst 33342-stained cells (blue fluorescence). (D) Representative results for tumor weight changes after subcutaneous injection of H1299 cells. Photographs show tumor xenograft morphologies in the LV-SMP30 + LV-HDAC4 and LV-SMP30 + LV-control H1299 groups. (E) Representative Western blot results of SMP30, HDAC4, c-Myc and CyclinD1 in H1299 xenograft tumor tissues. β-actin was used as a Western blot loading control. The values below the Western blot band represent the relative gray values. All the data are shown as the mean ± SD. Student’s t-test, *, P<0.05 vs. the LV-SMP30 control group; #, P<0.05 vs. the LV-SMP30 group; &, P<0.05 vs. the LV-SMP30 control + LV-HDAC4 group. HDAC, histone deacetylase; LV, lentivirus; SMP30, senescence marker protein 30; SD, standard deviation; NSCLC, non-small cell lung cancer.

Article Snippet: Antibodies against SMP30 (1:1,000, 17947-1-AP, Proteintech), against HDAC4 (1:1,000, Proteintech Cat# 17449-1-AP, RRID: AB_2118864), anti- CyclinD1 (1:1,000, 60186-1-Ap, Proteintech), anti- c-Myc (1:1,000, #5605, CST) anti- β - actin (1:5,000, ab6276, Abcam), and anti- tubulin (1:1,000, #2148, CST) were used.

Techniques: Western Blot, Colony Assay, Staining, Fluorescence, Injection, Control, Histone Deacetylase Assay, Marker, Standard Deviation

Journal: Translational Lung Cancer Research

Article Title: Senescence marker protein 30 inhibits tumor growth by reducing HDAC4 expression in non-small cell lung cancer

doi: 10.21037/tlcr-21-982

Figure Lengend Snippet: Effect of pharmacologic inhibition of HDAC4 on proliferative abilities of H1299 cells. LV-control and LV-HDAC4 H1299 cells were pretreated with SAHA or TMP269 for 48 h. (A,B) Representative Western blot results of HDAC4, c-Myc, and CyclinD1. (C,D) Representative images and results of colony formation assay. Colonies were visualized by crystal violet staining. (E,F) Representative images (×20) and statistical analysis of EdU incorporation assay. The results are presented as the ratio between the number of EdU-positive cells (red fluorescence) and the total number of Hoechst 33342-stained cells (blue fluorescence). *, P<0.05 vs. the LV-control +SAHA/TMP269 0 µM group; #, P<0.05 vs. the LV-control + SAHA1.25 µM/TMP269 40 µM group; &, P<0.05 vs. the LV-HDAC4 + SAHA/TMP269 0 µM group. HDAC, histone deacetylase; LV, lentivirus; SMP30, senescence marker protein 30.

Article Snippet: Antibodies against SMP30 (1:1,000, 17947-1-AP, Proteintech), against HDAC4 (1:1,000, Proteintech Cat# 17449-1-AP, RRID: AB_2118864), anti- CyclinD1 (1:1,000, 60186-1-Ap, Proteintech), anti- c-Myc (1:1,000, #5605, CST) anti- β - actin (1:5,000, ab6276, Abcam), and anti- tubulin (1:1,000, #2148, CST) were used.

Techniques: Inhibition, Control, Western Blot, Colony Assay, Staining, Fluorescence, Histone Deacetylase Assay, Marker

Journal: Nature Communications

Article Title: IL6 supports long-term expansion of hepatocytes in vitro

doi: 10.1038/s41467-022-35167-8

Figure Lengend Snippet: a Schematic of IL6-induced hepatocyte to iHPC reprogramming and subsequent maturation to iMHs. b Representative images of the primary hepatocytes (Hepa, D0) and hepatocytes cultured with or without IL6 for 14 days (D14) (repeated for three times). c Growth curves of primary hepatocytes cultured with or without IL6 for 14 days. Data are Means ± SEM ( n = 3 independent experiments). *** p < 0.001 (two-way ANOVA). Immunofluorescence staining of cell cycle markers CyclinD1 (red) and Ki67 (green), HPC markers Sox9 (red) and Ck19 (green) ( d ) and hepatic markers Hnf4α (red) Albumin (green), Cyp1a2 (red) and Cyp2c9 (green) ( e ) in primary hepatocytes (Hepa), IL6-iHPCs (D14) and IL6-iMHs (D21). Nuclei were stained with Hoechst 33342 (blue). f Statistical data of the immunofluorescence staining data in d and e . Data are Means ± SEM (Cells from three mice were analyzed independently, three random fields/mouse). * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t test). g Representative images of PAS staining and ICG uptake in primary hepatocytes and IL6-iMHs (D21) (repeated for three times). Scale bars represent 100 µm. DEX dexamethasone, OSM oncostatin M. Source data and the exact p values are provided in a Source data file.

Article Snippet: The following antibodies were used in this study: CyclinD1 (1:200; Abcam ab134175), Ki67 (1:200; Cell Signaling Technology 9129S), Sox9 (1:200; Millipore ab5535), Cytokeratin 19 (1:200; Abcam ab52625), Albumin (1:200; Bethyl, A90-234A), Hnf4α (1:200; Abcam ab181604), Cyp1a2 (1:200; Abcam ab22717), Cyp2c9 (1:200; Abcam ab4236), Fah (1:200; GeneTex GTX114400), GS (1:500; Abcam, ab49873), Arg1 (1:200; Abcam, ab96183).

Techniques: Cell Culture, Immunofluorescence, Staining, Two Tailed Test