Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: siRNA screen to identify Rab proteins affecting cell migration (A) U2OS cells transfected with control siRNA or pools of four different siRNAs against human Rabs were grown to confluency, scratch-wounded, and imaged every 3 h. The graph shows the relative wound density at 18 h after wounding represented as mean ± SEM of four independent experiments. The red solid line indicates the relative wound density (%) and the dashed red lines the boundaries of the SEM for cells transfected with control siRNA. (B) Representative images are shown for time 0 and 18 h after wounding for the two Rabs whose depletion had the strongest effect on wound closure. Scale bar: 200 μm. (C) U2OS cells transfected with control siRNA or a pool of four different siRNAs targeting Rab33b were imaged for 48 h. The rate of proliferation (measured as percentage of cell confluence) over time is shown as mean ± SEM of three independent experiments. (D) U2OS cells were transfected with control siRNA or each of the different individual four siRNAs present in the pool targeting Rab33b (Rab33b siRNA_1, siRNA_2, siRNA_3, or siRNA_4), grown to confluency, scratch-wounded, and imaged every 3 h. Representative images for time 0 and 24 h after wounding are shown. Scale bar: 200 μm. (E) Graph showing the relative wound density (%) over time for each sample in (d). The graph represents the mean ± SEM of a minimum of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, n.s. not significant, for t = 24h (two-tailed paired Student′s t-test). (F) Lysates from U2OS cells transfected with control siRNA, or with each of the different individual four siRNAs present in the pool targeting Rab33b were subjected to Western blot analysis with the indicated antibodies.

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Migration, Transfection, Two Tailed Test, Western Blot

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: Rab33b depletion promotes cell migration (A) U2OS cells transfected with either control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b or transiently transfected with GFP, or GFP-Rab33b T47N, were scratch-wounded and imaged every 3 h. Representative images for time 0 and 23 h after wounding are shown. Scale bar: 200 μm. (B) Graph showing relative wound density (%) for each sample in (a) over time. The graph represents the mean ± SEM of a minimum of three independent experiments. ∗p < 0.05, n.s., not significant, for t = 24h (two-tailed paired Student′s t-test). (C) Cell lysates from cells treated with control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, or treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b were subjected to Western blot analysis with antibodies against Rab33b and tubulin (as loading control). (D) Representative track plots of the single-cell distances of migration for cells transfected with control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, or treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b. Individual tracks are shown so that each starts at the origin (distance 0). (E) Quantification of the mean ± SEM of the single cell speed from at least three independent experiments. n > 30 cells per condition and per experiment. ∗p < 0.05, ∗∗p < 0.01, n.s., not significant (two-tailed paired Student′s t-test). See also Figures S1 A–S1B

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Migration, Transfection, Two Tailed Test, Western Blot

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: Rab33b is involved in the regulation of focal adhesion dynamics (A) U2OS cells silenced with control siRNA, Rab33b siRNA_1, or Rab33b siRNA_2, were fixed and stained with DAPI, rhodamine-conjugated phalloidin, and an antibody against vinculin. Scale bar: 5 μm. (B–C) Quantification of FA number per 100 μm 2 cell area (b) and size (c). The graphs represent the mean ± SEM for three independent experiments (n > 90 cells). ∗p < 0.05, n.s., not significant (two-tailed paired Student′s t-test). (D) Control or Rab33b-depleted cells transfected with RFP-vinculin were imaged every 10 min for 40 min. Arrows show FA disassembly, and arrowheads show FA assembly. Scale bar: 10 μm. (E) Rainbow color representation of FA assembly and disassembly over time from cells shown in panel (d). Each time point is shown in a different color, as indicated in the bar. Insets show magnifications of the boxed areas. Scale bar: 10 μm. (F) Quantification of assembly and disassembly rates of FAs. The assembly and disassembly rate is shown as percentage of focal adhesion formation or disassembly per minute. The values represent the mean ± SEM from three independent experiments, in which 15 FAs were analyzed per cell (n > 5), per condition, and per experiment. ∗p < 0.05, n.s., not significant (two-tailed paired Student′s t-test). (G) Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and RFP-vinculin (red). Cells were imaged every 5 s with a Zeiss LSM880 confocal microscope. Magnifications of the boxed areas in the side panels show Rab33b-positive vesicles moving to focal adhesion sites. Scale bar: 5μm, inset: 1μm. See also Figure S2 and Video S1. Rab33b-positive vesicles are delivered to FAs, Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and vinculin-RFP (red). Magnification of the boxed area 1 in Figure 4g is shown and illustrates two examples of Rab33b-positive vesicles contacting focal adhesions. Cells were imaged every 5 s using a spinning disk confocal microscope. Related to Figure 4. , Video S2. Rab33b-positive vesicles are delivered to FAs,Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and vinculin-RFP (red). Magnifications of the boxed area 2 in Figure 4g is shown and illustrate two examples of Rab33b-positive vesicles contacting focal adhesions. Cells were imaged every 5 s using a spinning disk confocal microscope. Related to Figure 4. , Video S3. Rab33b-positive vesicles are delivered to growing FAs during membrane protrusion,U2OS cells transiently transfected with GFP-Rab33b (green) and vinculin-RFP (red) were imaged every 30 s using a TIRF microscope with a penetration depth of 90 nm. The movie shows the recruitment of GFP-Rab33b-positive vesicles during membrane protrusion. Related to Figure 4. .

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Staining, Two Tailed Test, Transfection, Live Cell Imaging, Microscopy

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: VSV-G transport to the cell surface is inhibited by Rab33b depletion (A) U2OS cells silenced with control siRNA, siRNA against Rab33b, or silenced with Rab33b siRNA and transfected with RFP-Rab33b (red), were transfected with YFP-VSV-G (green) and incubated at 39°C for 16 h. Cells were then fixed either immediately (T0), 20 min (T20), or 90 min (T90) after a shift to 32°C. Scale bar: 10 μm. (B) Quantification of the VSV-G distribution 90 min after the shift to 32°C. 200 cells were analyzed from three independent experiments and the percentage of cells in which YFP-VSV-G was located at the Golgi, post-Golgi vesicles, and plasma membrane was determined. The graph shows the mean ± SEM ∗p < 0.05, ∗∗p < 0.01, n.s., not significant (two-tailed paired Student′s t-test). See also Figure S4 .

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Transfection, Incubation, Two Tailed Test

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet:

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Transduction, Recombinant, Two Hybrid Screening, Sequencing, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer

doi: 10.1101/2023.03.19.533333

Figure Lengend Snippet: ( A ) Oxygen tension (y-axis) as determined by an OxyLite probe in tumors from KPC mice breathing ambient air or pure oxygen (x-axis). Dark blue circles represent averages per tumor and boxplots illustrate their distribution. Light blue circles represent repeat measurments per tumor. ( B ) HIF target genes (orange) controlled directly or indirectly by BMAL2 (yellow). Indirect control involves both BMAL2’s negative influence on first (dark blue) or second tier (light blue) RP repressing HIF target genes, and positive influence on first (dark red) and second (light red) tier RP activating HIF target genes. ( C ) Fold change in cell growth relative to cells expressing non-targeting sgRNA in normoxic (21% O2) and hypoxic (1% O2) conditions ( D ) Number of migrated cells for cells expressing non-targeting or BMAL2-directed sgRNA in the indicated oxygen environment. P-values are derived from testing the indicated coefficients from a linear regression model. Significant interaction term suggests synergistic effects of BMAL2 pertubation and hypoxia on cell migration. ( E ) Media pH levels after 72 hours incubation. P-values derived from Welch’s t-test ( F ) Fold change in lactate levels in the indicated oxygen environment expressing either non-targeting or BMAL2-directed sgRNA ( G ) Western Blot for HIF1a, HIF2a,

Article Snippet: The sgRNA (small-guide RNA) to knocking-out BMAL2 as well as non-targeting (NT) sequence were purchased from GenScript (Piscataway, NJ) using pLentiGuide-Puro vector as a backbone.

Techniques: Control, Expressing, Derivative Assay, Migration, Incubation, Western Blot

Journal: Heliyon

Article Title: Long non-coding RNA TTTY14 promotes cell proliferation and functions as a prognostic biomarker in testicular germ cell tumor

doi: 10.1016/j.heliyon.2023.e16082

Figure Lengend Snippet: Expression of Testis-specific transcript, Y-linked 14 (TTTY14). (A) GEPIA was used to analyze the expression of TTTY14 in various tumors. TTTY14 is highly expressed in GBM, PRAD and TGCT. The normalization method is Transcripts Per Million (TPM). The scale of y-axis is log2(TPM+1). (B) The expression of TTTY14 in normal testis samples and testicular tumor samples was analyzed using the GSE3218 dataset in the GEO database. (C) The expression of TTTY14 in non-seminoma and seminoma was analyzed by GEPIA. (D) The TGCT cohort data was downloaded from the UCSC XENA database to analyze the expression of TTTY14 in intratubular germ cell tumors. *P < 0.05, **P < 0.01.

Article Snippet: The TTTY14 siRNA sequences and non-target negative control (NC) was synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing

Journal: Heliyon

Article Title: Long non-coding RNA TTTY14 promotes cell proliferation and functions as a prognostic biomarker in testicular germ cell tumor

doi: 10.1016/j.heliyon.2023.e16082

Figure Lengend Snippet: The association between TTTY14 expression, methylation, and copy number in TCGA TGCT cohort. The UCSC XENA database was used to download the methylation data, expression data, and prognosis data of TTTY14 from the TCGA TGCT cohort. (A–B) The copy number was significantly positively correlated with TTTY14 expression. The copy number column was sorted from smallest to largest values. The redder the color, the larger the value; the bluer the value, smaller the value. (C) Correlation between TTTY14 copy number and overall survival of TGCT cohort from TCGA. Patients with high TTTY14 copy number had lower survival probability. (D) The CpG sites of TTTY14 were showed. (E–F) TTTY14 methylation level was significantly negatively correlated with its expression. The DNA methylation column was sorted from smallest to largest values. The redder the color, the larger the value; the bluer the value, smaller the value. CNV, copy number various. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The TTTY14 siRNA sequences and non-target negative control (NC) was synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing, Methylation, DNA Methylation Assay

Journal: Heliyon

Article Title: Long non-coding RNA TTTY14 promotes cell proliferation and functions as a prognostic biomarker in testicular germ cell tumor

doi: 10.1016/j.heliyon.2023.e16082

Figure Lengend Snippet: Relationship between TTTY14 and survival prognosis of TGCT patients. Based on TCGA TGCT data, the Kaplan-Meier Plotter online tool was used to analyze relationship between TTTY14 expression and two-year survival in TGCT patients. (A) The relationship between TTTY14 expression and two-year survival in all TGCT patients was analyzed. (B) The relationship between TTTY14 expression and two-year survival in TGCT patients with high tumor mutational burden (TMB) was analyzed. (C) The relationship between TTTY14 expression and two-year survival in TGCT patients with low TMB was analyzed. (D) The relationship between TTTY14 expression and two-year survival in CD8 + T cell-enriched TGCT patients was analyzed. (E) The relationship between TTTY14 expression and two-year survival in patients with TGCT CD8 + T cell-deserted was analyzed.

Article Snippet: The TTTY14 siRNA sequences and non-target negative control (NC) was synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing

Journal: Heliyon

Article Title: Long non-coding RNA TTTY14 promotes cell proliferation and functions as a prognostic biomarker in testicular germ cell tumor

doi: 10.1016/j.heliyon.2023.e16082

Figure Lengend Snippet: Knockdown of TTTY14 inhibits NCCIT cell proliferation. (A) NCCIT cells were transfected with TTTY14 siRNA, and the expression of TTTY14 was detected by qPCR. (B) Edu staining was used to measure the proliferation of NCCIT cells transfected with TTTY14 siRNA. (C) CCK8 assay for cell viability of NCCIT cells transfected with TTTY14 siRNA. (D) Colony formation assay was used to detect the cell cloning ability of NCCIT cells transfected with TTTY14 siRNA. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The TTTY14 siRNA sequences and non-target negative control (NC) was synthesized by Ribobio (Guangzhou, China).

Techniques: Knockdown, Transfection, Expressing, Staining, CCK-8 Assay, Colony Assay, Cloning

Journal: Heliyon

Article Title: Long non-coding RNA TTTY14 promotes cell proliferation and functions as a prognostic biomarker in testicular germ cell tumor

doi: 10.1016/j.heliyon.2023.e16082

Figure Lengend Snippet: The correlation between TTTY14 and antitumor drug sensitivity. GSCA online tool was used to analyze the correlation between TTTY14 expression and the sensitivity of Genomics of Drug Sensitivity in Cancer (GDSC) drugs (top 30) in TGCT (A), and the correlation between TTTY14 expression and the sensitivity of cancer therapeutics response portal (CTRP) drugs (top 30) in TGCT (B). (C) Based on anti -PD1 cohort data, the Kaplan-Meier Plotter online tool was used to analyze the correlation between TTTY14 expression and the immune checkpoint inhibitors. Relationship between overall survival and progression-free survival in this cohort of patients was shown. High expression of TTTY14 is a predictive marker for poor outcome of anti -PD1immunotherapy in TGCT patients.

Article Snippet: The TTTY14 siRNA sequences and non-target negative control (NC) was synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing, Marker

Journal: Heliyon

Article Title: Long non-coding RNA TTTY14 promotes cell proliferation and functions as a prognostic biomarker in testicular germ cell tumor

doi: 10.1016/j.heliyon.2023.e16082

Figure Lengend Snippet: The TTTY14-associated genes, proteins, and pathways. Based on the TCGA TGCT expression profile data, LinkedOmics was used to analyze the positively associated genes and negatively associated genes of TTTY14 (A), and the associated proteins of TTTY14 (B). (C) GSEA enrichment was performed to analyze the associated pathway of TTTY14.

Article Snippet: The TTTY14 siRNA sequences and non-target negative control (NC) was synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing

Journal: Heliyon

Article Title: Long non-coding RNA TTTY14 promotes cell proliferation and functions as a prognostic biomarker in testicular germ cell tumor

doi: 10.1016/j.heliyon.2023.e16082

Figure Lengend Snippet: Relationship between TTTY14 expression, immune cells and immune checkpoint molecules. Based on the TCGA TGCT cohort data, the ASSISTANT for Clinical Bioinformatics tool was used to analyze the abundance of various immune cells in TGCT samples (A), the correlation between TTTY14 expression and immune dysregulation score (B), and the correlation between TTTY14 and the abundance of various immune cells (C). (D) The correlation between TTTY14 and three immune checkpoint molecules CD274, CTLA4 and HAVCR2 was analyzed using the GEPIA database.

Article Snippet: The TTTY14 siRNA sequences and non-target negative control (NC) was synthesized by Ribobio (Guangzhou, China).

Techniques: Expressing