MOESM1 of Engineering Bacillus licheniformis as a thermophilic platform for the production of l-lactic acid from lignocellulose-derived sugars

Additional file 1: Figure S1. The construction of gene knockout plasmids. Figure S2. The analysis of the stereoisomers of lactic acid produced by strain RH02. Table S1. Primers used in this study. Text 1. The codon-optimized sequences of xylAs

The JC-1 staining of mitochondria (A) and co-localization of mitochondria with cytochrome c (B and C) in apoptosis induced with AcMNPV in SL-HP cells under amino acid deprivation.

<p>(A1) SL-HP cells starved for 6.5 h, (A2) SL-HP cells infected for 5 h under starvation (6.5 h), showing damaged mitochondria with yellow fluorescence, (A3) unstarved SL-HP cells, (B) SL-HP cells starved for 6.5 h showing the co-localization of cytochrome <i>c</i> with mitochondria, (C) SL-HP cells infected for 5 h under starvation (6.5 h); no co-localization of cytochrome c with mitochondria was observed. Arrow pointed to damaged mitochondria. Bars = 20 µm.</p

An increase in autophagy activity in AcMNPV-infected SL-HP cells in comparison with non-infected SL-HP cells under starvation pressure.

<p>(A) Distribution of GFP-HaAtg8 punctual spots in non-infected SL-HP cells starved for 4.5 h, (B) Distribution of GFP-HaAtg8 punctual spots in AcMNPV-infected SL-HP cells starved for 4.5 h at 2 h of post-infection, showing the increase of autophagosomes in comparison with starved SL-HP cells, (C) Assay of acid phosphatase (ACP) activity between non-infected and infected SL-HP cells starved for 6 h at 4 h of post-infection.</p

PCR primers used in this study.

a<p>: The lower-case letters indicate the corresponding protective bases of the restriction enzymes. The underlining letters indicate the cleavage sites of the restriction enzymes.</p

An increase of autophagy activity in starved SL-HP cells.

<p>(A) Distribution of GFP-HaAtg8 punctual spots in unstarved SL-HP cells, (B) Distribution of GFP-HaAtg8 punctual spots in SL-HP cells starved for 2 h, showing the increase of autophagosomes in comparison with unstarved SL-HP cells, (C) Acid phosphatase (ACP) activity in SL-HP cells starved for different periods. Bars = 15 µm.</p

A shift from autophagy to apoptosis triggered by baculovirus under amino acid deprivation.

<p>The starved SL-HP cells (A1–3) and the infected cells under starvation (B1–3) were shown. (A1) Observation of the cells starved for 48 h using light microscope, (A2) Hoechst staining of the cells starved for 48 h; (A3) DNA agarose electrophoresis showing that no DNA degradation occurred after 0, 24, 48 and 72 h of starvation. (B1) Observation of the cells infected for 24 h using light microscope, (B2) Hoechst staining of the cells infected for 24 h under starvation (25.5 h), (B3) DNA agrose electrophoresis showing that DNA fragmentation occurred in AcMNPV-infected SL-HP cells under starvation at 12 h or 24 h of post-infection, (C1) unstarved SL-HP cells, (C2) unstarved SL-HP cells stained with Hoechst; (C3) DNA extracted from SL-HP cells starved for 24 h was run at 1×, 2× and 4×; (D) Analysis of relative caspase-3 activity, (D1) Starved cells; (D2) AcMNPV-infected cells under starvation condition. *<i>p</i><0.05. Bars = 20 µm. M, 100 bp DNA ladder.</p

Replication of four baculoviruses in <i>S. litura</i> SL-HP cells cultured in complete medium with 10% FBS.

<p>(A) AcMNPV infection for 48 h, (B) Ac-PH-GFP-actin infection for 48 h, (C) AfMNPV infection for 48 h, (D) SplitMNPV infection for 72 h, (E)Unstarved cells, (F) Starvation for 24 h, (G) Relative caspase 3 activity in AcMNPV-infected SL-HP cells at different time points of post-infection, (H) DNA ladder assay for AcMNPV-infected SL-HP cells at different time points of post-infection (0, 12, 24, 36, 48 h). Bars = 20 µm.</p

The expression, localization, abundance and cleavage of Atg6 in insect cells.

<p>(A) the purified BmAtg6-His/V5 expressed in <i>E.coli</i>, (B) LD652 cells transfected with plasmid BmAtg6-GFP and observed with the confocal laser microscopy at 24 h post transfection, showing the localization of over-expressed BmAtg6-GFP protein; The blue light emitted by Hoechst was changed to red light with software, (C) localization of the endogenous SlAtg6 in SL-HP cells shown by immunofluorescence using anti-BmAtg6 mouse serum. (D)Abundance alteration of SlAtg6 under various conditions; lane 1, unstarved Sl-HP cells; lane 2, SL-HP cells starved for 6 h; lane 3, AcMNPV-infected SL-HP cells at 4 h of post-infection; lane 4, AcMNPV-infected SL-HP cells starved for 6 h at 4 h of post-infection, (E) No cleavage of endogenous SlAtg6 was observed in normal SL-HP cells (control) and apoptotic cells induced with actinomycin D (1 µg/ml) by western blot using anti-BmAtg6 serum. Bars = 20 µm.</p

The expression analysis of <i>p35</i> and <i>ie-1</i> genes from AcMNPV using semi-q-PCR in unstarved and starved SL-HP cells, respectively.

<p>CV: cells cultured in complete medium with 10% FBS and infected for 6 h; SV: cells infected for 6 h under starvation (7.5 h), and <i>actin3A</i> gene was used as internal control.</p

Cytochrome c dsRNA silenced cytochrome c expression in Sl-1 cells.

<p>(A) Identification of recombinant plasmid pLitmus-cytc containing cytochrome c DNA fragment. pLitmus-cytc was digested with BamH I and EcoR I and then analyzed by agarose gel electrophoresis. M1: 1 kb maker, M2: 100 bp maker, S: plasmid DNA products. (B) Electrophoretic analysis of dsRNA transcribed <i>in vitro</i>. pLitmus-cyt c was digested with BamH I and EcoR I respectively and transcribed <i>in vitro</i>, and RNA products were electrophorised on agrose gel. M: 100 bp maker, S: RNA transcription products. (C) Cytochrome c mRNA level after treatment with dsRNA. Sl-1 cells treated with dsRNA for 0, 24, 48, and 72 h, and after inoculated with AfMNPV for 10 h, total RNA in each treatment was isolated. Cytochrome c mRNA was determined by semi-quantitative RT-PCR.</p