Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: a The relative expression of LINC00973 in normal human oral keratinocytes (HOK) cell and HNSCC cell lines were measured by qRT-PCR. b Subcellular RNA fraction and followed qRT-PCR assays were conducted to determine the localization of LINC00973 within Cal27 and HN6 cells. GAPDH and U6 served as the internal control of cytoplasm and nuclear, respectively. c RNA FISH assays showed LINC00973 mainly localized in cytoplasm in Cal27 and HN6 cells. Scale bar: 10 μm. d The knockdown efficiency of LINC00973 was determined by qRT-PCR in Cal27 and HN6 cells transfected si-LINC00973-1 (si-1) and si-LINC00973-2 (si-2). Cell proliferation, migration, and invasion was evaluated by CCK-8 ( e ), wound-healing ( f ), and Transwell invasion assays ( g ) in LINC00973-sliencing Cal27 and HN6 cells. Scale bar: 50 μm. h The protein abundance of indicated migration and invasion related markers was detected in LINC00973-deficient cells. i Schematic description of experimental procedures for the orthotopic floor-of-mouth xenograft model. j Representative image of the tumor masses with stable LINC00973 knockdown/control HN6 cells and the final volume of samples were recorded. Scale bar: 1 cm. k Representative IHC staining of Ki67 in xenograft tumors (left panel) and their quantification data were shown (right panel). Scale bar: 50 μm. l Representative H&E staining and IHC staining of CK5/6 in lymph nodes (left panel) from orthotopic tumor-bearing mice. The percentage of lymph nodes metastasis were shown and compared (right panel). Fisher’s exact test. Data shown here are mean ± SD from three independent experiments, ** P < 0.01, Student’s t or one-way ANOVA test.

Article Snippet: After blocking with QuickBlock Blocking Buffer (#P0220, Beyotime, China), sections were incubated overnight at 4 °C with primary antibodies against CK5/6 (#MAB-0744, Maixin, China), Ki67 (#MAB-0672, Maixin, China), EN2 (1:100, #sc-293311, Santa Cruz, USA), FOSL1 (1:1000, #5281, Cell signaling, USA) or NOTCH1 (1:500, #20687-1-AP, Proteintech, China).

Techniques: Expressing, Quantitative RT-PCR, Control, Knockdown, Transfection, Migration, CCK-8 Assay, Quantitative Proteomics, Immunohistochemistry, Staining

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: Heatmap ( a ) and volcano plot ( b ) displayed the expression profile of DEGs following LINC00973 knockdown or control cells. The mRNA ( c ) and protein ( d ) expression of EN2 were measured in Cal27 and HN6 cells with/without EN2 knockdown. e , f The correlations between EN2 and LINC00973 RNA expression were assessed in the public HNSCC datasets ( GSE41613 and GSE42743 ) and our in-house samples. Spearman’s correlation. The mRNA ( g ) and protein ( h ) abundance of EN2 were measured in Cal27 and HN6 cells under four conditions. Cell proliferation, migration and invasion were significantly reduced following LINC00973 knockdown but restored by ectopic EN2 overexpression as gauged by CCK-8 ( i ), wound healing ( j ) and Transwell invasion assays ( k ). Scale bar: 50 μm. l The migration/invasion-related markers were determined by western blot in Cal27 and HN6 cells with/without LINC00973 knockdown coupled with/without EN2 overexpression. m 1 × 10 5 HN6 cells with LINC00973 or/and EN2 manipulations were injected submucosally into the floor of the mouth. Fourteen days post-injection, tumor masses and cervical lymph nodes were harvested. Representative tumor masses images and estimated tumor volumes were showed. Scale bar: 1 cm. Representative IHC staining of Ki67 and EN2 in xenograft tumors ( n ) and their quantification data were shown ( o ). Scale bar: 50 μm. p Representative IHC staining of CK5/6 in lymph nodes (left panel) from orthotopic tumor-bearing mice among four indicated groups were displayed. The percentage of lymph nodes metastasis were shown and compared (right panel). Fisher’s exact test. Data were presented as mean ± SD, ** P < 0.01, Student’s t test.

Article Snippet: After blocking with QuickBlock Blocking Buffer (#P0220, Beyotime, China), sections were incubated overnight at 4 °C with primary antibodies against CK5/6 (#MAB-0744, Maixin, China), Ki67 (#MAB-0672, Maixin, China), EN2 (1:100, #sc-293311, Santa Cruz, USA), FOSL1 (1:1000, #5281, Cell signaling, USA) or NOTCH1 (1:500, #20687-1-AP, Proteintech, China).

Techniques: Expressing, Knockdown, Control, RNA Expression, Migration, Over Expression, CCK-8 Assay, Western Blot, Injection, Immunohistochemistry

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A and B , High (A, KRT5+, >40% stained neoplastic cells, n = 37) and low (B, KRT5-, n = 160) frequency of KRT5 expressing cancer cells. C , Kaplan-Meier survival analysis of HGSC patients stratified according to frequency of KRT5 expressing cells (P=0.0015). A and B, Elite ABC method. Hematoxylin counterstaining. Scale bar, 100 µm.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A , Structure of Lenti-KRT5mCherry (L-KRT5mCherry). B , Co-localization of KRT5 immunostaining (KRT5, green) and mCherry (L-KRT5) expression (magenta). Orange, overlay. Counterstaining with DAPI (blue). Scale bar, 60 µm. C , Quantification of cells expressing both KRT5 and L-KRT5 (KRT5+ L-KRT5+), or only KRT5 (KRT5+) or L-KRT5+. D , Experimental design of isolation of cells expressing both L-KRT5mCherry and L-hUbC-GFP or L-hUbC-GFP alone. E, PCR detection of L-hUbC-GFP (GFP) and L-KRT5mCherry (mCherry) DNA in both KRT5+ (K5+) and KRT5- (K5-) cells. All error bars denote s.d.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Immunostaining, Expressing, Isolation

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A and B , Organoids derived from SKOV3 cells expressing both lentiviruses (A, GFP and mCherry, orange) or GFP alone (B, green). Scale bar, 100 µm. C , Quantification of KRT5+ (blue symbols, pink bars) and KRT5- (pink symbols, yellow bars) cancer organoids in 6 consecutive passages. All error bars denote s.d. D, Volume of tumors formed by serially diluted (1 x 10 5 , 1 x 10 4 , 1 x 10 3 ) of KRT5+ and KRT5- cells after their s.c. transplantation into different flanks of NSG mice. KRT5-group did not form tumors. E and F , mCherry (E) and KRT5 (F) expression in KRT5+ cell derived xenografts. Elite ABC method. Hematoxylin counterstaining. Scale bar, 60 µm. All error bars denote s.d. G. Live microscopy of cells were isolated by FACS based on their expression of GFP (green) and mCherry (magenta) after coinfection with Lenti-UbC-GFP and Lenti-KRT5mCherry. Orange, Overlay. Individual frames of live microscopy. Scale bar, 60 µm.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Derivative Assay, Expressing, Transplantation Assay, Microscopy, Isolation

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A. Effect of cisplatin and doxorubicin on KRT5+ and KRT5- cells. Manual count (cell viability, upper row) and MTT assay (absorbance at 590 nm, bottom row) of KRT5+ and KRT5- cells after 72 hours of treating with cisplatin and doxorubicin at different concentrations. B and C . Representative confocal images of primary HGSC organoids (B) and quantification of organoid frequency (B, left image) and size (C, right image) after treatment with different concentrations of cisplatin for 72 hours. C, Immunofluorescence for KRT5 (green). Counterstaining with DAPI (blue). Scale bar, 50 µm for all images. All error bars denote s.d.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: MTT Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A . Heat map depicting the top 200 differentially expressed genes in KRT5+ (A2, A1 and A4) and KRT5- (A7, A6, and A5) cells. Note the expression of SPP1 (osteopontin) in KRT5- cells (arrow). B and C . DAVID analysis for functional annotation of terms associated with KRT5+ (B) and KRT5- (C) cells.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Expressing, Functional Assay

Journal: bioRxiv

Article Title: Keratin 5 marks cancer-propagating cells sustained by an osteopontin-producing niche in high-grade serous ovarian carcinoma

doi: 10.64898/2026.01.28.702332

Figure Lengend Snippet: A and B. Quantification of cells expressing KRT5 and/or SPP1 cells within human HGSC cases from single-cell RNA sequencing. Significance by Mann-Whitney U test. C. RT-PCR analysis of SPP1 expression in KRT5+ and KRT5- subpopulations of SKOV3 cells. D and E, KRT5 (green) and OPN (red) expression in HGSC (D) and primary HGSC organoid (E). Double immunofluorescence, counterstaining with DAPI (blue). Scale bar, (D) 60 µm, E (40 µm). F and G , OPN treatment increases frequency (F) and size (G) of HGSC organoids (n=3). H - K . Effect of cisplatin on frequency (H and J) and size (I and K) of organoids either transduced with SPP1 shRNA (H and I) or treated with OPN (I and K). All organoids were measured 72 hours after treating with cisplatin at different concentrations. All error bars denote s.d.

Article Snippet: Briefly, for lentivirus packaging psPAX2 (Addgene, 12260), pMD2.G envelope plasmid (Addgene, 12259), and KRT5 promoter clone (GeneCopoeia, HRPM15909-LvPM02, mCherry) were employed.

Techniques: Expressing, RNA Sequencing, MANN-WHITNEY, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Transduction, shRNA